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INTERNATIONAL CONFERENCE ON ANAEROBIC PROTISTS

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INTERNATIONAL CONFERENCE ON ANAEROBIC PROTISTS Powered By Docstoc
					  INTERNATIONAL CONFERENCE ON ANAEROBIC PROTISTS
            Anaerobic protozoa: from basic science to useful applications
                                         Alghero, Italy
                                       September 17-20, 2005




        B22 EXPERT GROUP MEETING


Scientific Committee

Pier Luigi Fiori
University of Sassari, Italy
Graham Coombs
University of Glasgow, UK
Johannes Hackstein
Radboud University Nijmegen, The Netherlands
Yan Tachezy
Charles University, Czech Republic
Marcos Vannier dos Santos
University of Rio de Janeiro, Brazil
Nigel Yarlett (USA)
Pace University, New York, USA

Local Organizing committee

Paola Rappelli
University of Sassari, Italy

And
       Domenico Delogu
       Giuseppe Delogu
       Daniele Dessì
       Nicia Diaz
       Giovanna Sanciu



IN COOPERATION WITH

           The University of Sassari,
           CENTER FOR BIOTECHNOLOGY DEVELOPMENT AND BIODIVERSITY RESEARCH
           and
           DEPARTMENT OF BIOMEDICAL SCIENCES
                  COST ACTION B22
       DRUG DEVELOPMENT FOR PARASITIC DISEASES
               COST B22 WG2: Drug target characterisation




           COST B22
           Management Committee representative:

                             Professor Graham H. COOMBS
                                 Infection & Immunity
                                Joseph Black Building
                    University of Glasgow UK - G12 8QQ Glasgow
                           E-mail: g.coombs@bio.gla.ac.uk




                              ACKNOWLEDGMENTS

The organizers would like to acknowledge:

    BANCO DI SARDEGNA
    ERSU ENTE REGIONALE PER IL DIRITTO ALLO STUDIO
    FONDAZIONE BANCO DI SARDEGNA
    NUREX S.R.L.
    PRESIDENZA DEL CONSIGLIO REGIONALE DELLA SARDEGNA
    REGIONE AUTONOMA DELLA SARDEGNA
    UNIVERISITÀ DI SASSARI
INTERNATIONAL CONFERENCE ON ANAEROBIC PROTISTS
                     Anaerobic protozoa: from basic science to useful applications
                                  Alghero, Italy    September 17-20, 2005




                                               PROGRAM



                th
Saturday 17 September 2005

18.00   Registration at Chiostro di San Francesco
        Welcoming reception

           th
Sunday 18 September 2005

 9:00   Opening of the Meeting


        CELL BIOLOGY I
        Chairpersons: Johannes Hackstein and Marcos Vannier dos Santos

 9:00   Nancy Guillen (France): Myosin IB and cytoskeleton activities during phagocytosis in Entamoeba
        histolytica
 9:20   Marlene Benchimol (Brazil): Interaction of Trichomonas with bovine-cells of the reproductive tract
 9:40   Adrian Hehl (Switzerland): ESV organization and maturation in Giardia lamblia
10:00   Timothy Paget (UK): Apoptosis in Giardia and other anaerobic protozoans
10:20   Eva Nohynkova (Czech Republic): Cell division and flagellar developmental cycle of Giardia
10:40   Kevin Tan (Singapore): Recent advances in Blastocystis research – implications for protozoan
        programmed cell death and parasite survival


11:00   Coffee break


        CELL BIOLOGY II

11:15   Andreas Brune (Germany): "Endomicrobia": Cytoplasmic symbionts of termite gut protozoa form
        separate phylum of prokaryotes
11:35   Jan Tachezy (Czech Republic): Common mode of protein targeting into mitosomes and
        hydrogenosomes
11:55   Frances Gillin (USA): The centrosomes as a Giardia lamblia cellular control center in regulating
        differentiation
12:15   Jaroslav Kulda (Czech Republic): Assembly and disassembly of the adhesive disc during cell
        division of a wildtype and albendazole treated Giardia intestinalis
        MOLECULAR BIOLOGY I
        Chairpersons: Patricia Johnson and Jan Tachezy

14.30   Joana Silva (USA): The Trichomonas vaginalis genome sequencing project
14:50   Peter Upcroft (Australia): Genotyping Trichomonas vaginalis
15:10   Petrus Tang (Taiwan): Insights from the transcriptome of Trichomonas vaginalis

15:30   Jung-Hsiang Tai (Taiwan): A novel protein, Myb24, regulates transcription of the iron-inducible
        ap65-1 gene in the protozoan parasite Trichomonas vaginalis

15:50   Stepanka Vanacova (Switzerland): Spliceosomal introns in Trichomonas vaginalis
16:10   Tomoyoshi Nozaki (Japan): Comprehensive analysis of vesicular trafficking in Entamoeba
        histolytica


16.30   Coffee break


        MOLECULAR BIOLOGY II

16.45   Serge Ankri (Israel): Epigenetic regulation of gene expression in the protozoan parasite Entamoeba
        histolytica
17:05   Ching C. Wang (USA): Identification in Giardia lamblia of two eukaryotic translation initiation factor
        4E homologues with distinctive functions
17:25   Guenola Ricard (The Netherlands): More than 140 genes have been acquired through horizontal
        gene transfer from bacteria to rumen ciliates
17:45   Patricia Johnson (USA) Teamwork: Trichomonas vaginalis RNA polymerase II and the Initiator
        Binding Protein (IBP39)


18:15   Poster session

           th
Monday 19 September 2005

        HOST- PARASITE RELATIONSHIPS I
        Chairpersons: Michael Duchêne and Nigel Yarlett

9:15    Esther Orozco (Mexico): Entamoeba histolytica: EhDH112 is a novel member of the ALIX/AIP1
        protein family
9:35    David Mirelman (Israel): Entamoeba histolytica: epigenetic silencing of the amoebapore gene
        results in virulence – attenuated stable trophozoites
9:55    Michael Duchêne (Austria): A possible functional relationship between surface glycoproteins of
        Entamoeba histolytica and human phagocytes
10:15   John Alderete (USA): The Functional Interface Between Trichomonas vaginalis and its Host Cell
10:35   Patricia Johnson (USA) The role of cell surface glycoconjugates in the pathogenesis of
        Trichomonas vaginalis
10:55   Coffee break


        HOST- PARASITE RELATIONSHIPS II
11:10   Daniele Dessì (Italy): Trichomonas vaginalis and the first line of host defense
11:30   Robert Hirt (UK): Gene duplication and horizontal gene transfer in Trichomonas vaginalis genome
        evolution: identification of potential pathogenic factors
11:50   Rossana Arroyo (Mexico): Iron in the virulence of Trichomonas vaginalis
12:10   Emma Ringqvist (Sweden): Giardia - host cell interactions: it takes two to tango


        BIOCHEMISTRY
        Chairpersons: Louis Tielens and Ching C. Wang

14:00   Louis Tielens (The Netherlands): Energy generation in anaerobic protists
14:20   Johannes Hackstein (The Netherlands): Diversity and evolution of anaerobic energy-generating
        organelles
14:40   Ivan Hrdy (Czech Republic): Catalytic module of mitochondrial-type respiratory complex 1 in
        Trichomonas vaginalis hydrogenosomes
15:00   Siddhartha Das (USA): Ceramide endocytosis and metabolism by an amitochondriate protozoan,
        Giardia lamblia
15:20   Henning Scholze (Germany): The two inhibitors of cysteine proteases, amoebiasin-1 and –2, in
        trophozoites of Entamoeba histolytica
15:40   Nigel Yarlett (USA): Cryptosporidia infection alters host cell polyamine levels


20:30   Conference Dinner

            th
Tuesday 20 September 2005

        EVOLUTION
        Chairpersons: Andreas Brune and David Lloyd

9:00    David Lloyd (UK): The cardiolipin-containing mitochondria-like organelles of Giardia intestinalis
        (Syn. lamblia, duodenalis)
9:20    Eric Viscogliosi (France): Molecular phylogeny versus systematics based on morphological
        characters in parabasalids: conflict or congruence?
9:40    Neil McEwan (UK): The fibrolytic genes in rumen ciliates were acquired by lateral gene transfer
10:00   Jorge Tovar (UK): Giardia mitosomes: vestigial anaerobic mithocondria?
10:20   Robert Hirt (UK): Horizontal gene transfer in Entamoeba histolytica: evidence for an important role
        of recently acquired genes from prokaryotic origins


10:40   Coffee break
DRUGS AND DRUG TARGETS
        Chairperson: Jaroslav Kulda

11:00   Ed Jarroll (USA): Encystment as a target for drug design
11:20   Jacqui Upcroft (Australia): Kaletra – an effective anti-trichomonal and anti-giardial agent
11:40   Ching C. Wang (USA): Identification of a subversive substrate of Trichomonas vaginalis purine
        nucleoside phosphorylase and the crystal structure of the enzyme-substrate complex


        SELECTED ORAL PRESENTATIONS
        Chairpersons: Robert Hirt and Eric Viscogliosi


12:00   Vahab Ali et al. Characterization of cytosolic Fe-S cluster assembly under anaerobic conditions in
        Entamoeba histolytica
12:10   Katrin Henze et al. Putative [Fe]-hydrogenase maturases in the hydrogenosomes of Trichomonas
        vaginalis
12:20   David Leitsch et al. Reaction of Entamoeba histolytica to drugs and stress – a proteomic approach
12:30   Mary Morada et al. A new method for continuous culture of Cryptosporidium parvum
12:40   Shigeharu Moriya et al. Comparative EST analysis of symbiotic anaerobic protists of termite gut
12:50   Pavla Tumova et al. Karyotype and the course of mitosis in Giardia intestinalis


        CLOSING REMARKS
13:00   Piero Cappuccinelli (Italy): Health and social impact of infections due to anaerobic protozoa in
        developing countries
13:15   Closing of the meeting
                                        ORAL COMMUNICATIONS

1. Characterization of cytosolic Fe-S cluster assembly under anaerobic conditions in Entamoeba
    histolytica
    Vahab Ali, Kumiko Nakada-Tsukui, and Tomoyoshi Nozaki
    Dept. of Parasitology, Gunma University Graduate School of Medicine, Maebashi, Japan


2. Putative [Fe]-hydrogenase maturases in the hydrogenosomes of Trichomonas vaginalis
                  a                     b                          a           b              a
    Simone Pütz , Pavel Dolezal , Gabriel Gelius-Dietrich , Jan Tachezy and Katrin Henze
    a
    Institut für Botanik III, Heinrich Heine Universtität Düsseldorf, Universitätsstrasse 1, 40225
                                b
   Düsseldorf, Germany, Department of Parasitology, Faculty of Science, Charles University, Vinicna
   7, Prague 2 128 44, Czech Republic


3. Reaction of Entamoeba histolytica to drugs and stress – a proteomic approach
                      1             2                          1
    David Leitsch , Iain Wilson , and Michael Duchêne
    1
    Department of             Specific Prophylaxis and Tropical Medicine, Center for Physiology and
                                                                       2
   Pathophysiology, Kinderspitalg. 15, A-1095 Vienna, Austria; Department of Chemistry, Universität
   für Bodenkultur, Muthgasse 18, A-1190 Vienna


4. A new method for continuous culture of Cryptosporidium parvum
    Mary Morada* and Nigel Yarlett
    Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, New
   York, USA


5. Comparative EST analysis of symbiotic anaerobic protists of termite gut
                              1,3                    1,3                   2         1,2
    Shigeharu MORIYA , Nemuri TODAKA , Kanako SAITA , Moriya OHKUMA                           and Toshiaki
           1,3
    KUDO
    1                     2                                                    3
    RIKEN Institute, Japan Science and Technology Agency (JST), Graduate School of Yokohama
    City University


6. Karyotype and the course of mitosis in Giardia intestinalis.
                      1                     1              2
    Pavla Tumova , Eva Nohynkova and Jiri Kral
    1                                           st                                        2
    Department of Tropical Medicine, 1 Faculty of Medicine, Studnickova 7, Prague 2; Department of
    Genetics and Microbiology, Faculty of Science, Vinicna 5, Prague 2, Czech Republic.
                                                POSTERS
1. DOWN REGULATION BBY IRON OF THE CP65 CYSTEINE PROTEINASE INVOLVED IN THE
  TRICHOMONAS VAGINALIS CYTOTOXICITY TO HELA CELL MONOLAYERS
                        a,b                b
  Alvarez-Sánchez, ME         and Arroyo, R .
  a
      Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, México, D. F.,
           b
  México. Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados
  del I.P.N., Av. IPN # 2508, Col. San Pedro Zacatenco CP 07360, México, D.F., México.


2. PROTEIN PROFILE OF TRICHOMONAS VAGINALIS BY TWO-DIMENSIONAL POLYACRYLAMIDE
  GEL ELECTROPHORESIS
                                                             1
  Juan Pablo Cifuentes-Ortiz, Javier Ambrosio-Hernández , Fernando Anaya-Velázquez and Felipe
  Padilla-Vaca.
  Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
                              1
  Guanajuato, Gto. and        Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico
  City, México.


3. AN ARGININE-SPECIFIC MONO-ADP-RIBOSYL TRASFERASE ACTIVITY IN ENTAMOEBA
  HISTOLYTICA TROPHOZOITES
  Eva E. Avila, Susana ML Fuentes, Mónica E. Silva, Araceli López, Angel H. Alvarez and Guadalupe
  Martínez-Cadena
  Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
  P O Box 187, Guanajuato, Gto, México.


4. DNA    FROM     TRICHOMONAS           VAGINALIS   INDUCES         INOS    EXPRESSION   IN   MOUSE
  MACROPHAGES
  Patricia Cuéllar-Mata, E. Alderete-Ledezma, Martha Olivia Solís-Martínez and Sergio Arias-Negrete.
  Institute for Research in Experimental Biology and School of Chemistry, University of Guanajuato.
  Noria Alta s/n, col. Noria Alta, 36050, Guanajuato, Gto., Mexico.


5. DETECTION AND GENOTYPING OF GIARDIA DUODENALIS                           FROM HUMAN AND ANIMAL
  ISOLATES IN ITALY
   D. Di Cave1,2
  1 Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di Roma “Tor Vergata”, Rome, Italy.
  2 Azienda Ospedaliera Universitaria Policlinico “Tor Vergata”, Rome, Italy.


 6. CHARACTERISATION OF A DYNAMIN LIKE PROTEIN IN GIARDIA: GiDLP IS INVOLVED IN CYST
  FORMATION AND ENDOCYTOSIS
  Verena Gaechter and Adrian B. Hehl
  Institute of Parasitology, Winterthurerstr. 266a, CH-8057 Zürich
7. HUMAN sIgA DEGRADATION BY SURFACE PROTEASES OF ENTAMOEBA HISTOLYTICA.
   Rosa María García-Nieto, Sergio Arias-Negrete and Eva E. Avila.
   Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
   Noria Alta S/N, Col. Noria Alta, 36050, Guanajuato Gto., nietor@quijote.ugto.mx.


8. EFFECTS OF POLYAMINE DEPLETION BY DFMO ON HYDROGENOSOMES IN TRICHOMONAS
   VAGINALIS
                            1,2,3                       2                     1                         1                               1,2
   Kristina M. Harris                , Burt Goldberg , Michael Turner , Anthony Hayes , Katey M. Lemar , Nigel
           3                     1
   Yarlett , David Lloyd .
   1                                                                                                                             2
    Microbiology (Bios1), Cardiff University, Park Place, Cardiff CF10 3TL, Wales, UK                                             Department of
                                                                                                            3
   Chemistry, New York University, Silver Center, New York, NY 10003, USA Haskins Laboratories and
   Department of Chemistry and Physical Sciences, Pace University, 41 Park Row, New York, NY
   10038, USA


9. TRICHODB: A WEB-BASED RESOURCE FOR THE TRICHOMONAS VAGINALIS GENOME
                            1                       2                             3                                  3                         1
   Richard D. Hayes ; Jane M. Carlton ; Michael J. Cipriano ; Andrew G. McArthur ; Patricia J. Johnson
   1                                                                  2
       University of California – Los Angeles, CA, USA                    The Institute for Genomic Research, Rockland,
               3
   MD, USA Marine Biological Laboratory, Woods Hole, MA, USA


10. GENE       EXPRESSION                OF   ENTAMOEBA           HISTOLYTICA               UNDER               THE            INFLUENCE      OF
   METRONIDAZOLE AND ALKYLPHOSPHOCHOLINES
                   1                                2                     2                                      1
   Margit Hofer , Evelyn Hatzenbichler , Martin Schreiber , and Michael Duchêne
   1
    Department         of       Specific    Prophylaxis     and    Tropical           Medicine,       Center             for    Physiology    and
                            2
   Pathophysiology, Department of Obstetrics and Gynecology, Medical University Vienna, Austria


11. GENETIC DIFFERENCE OF E. HISTOLYTICA BY SUBTRACTIVE HYBRIDIZATION AND
   DIFFERENTIAL GENE EXPRESSION
   M. Pilar Crisóstomo-Vázquez, Enedina Jiménez-Cardoso
   Laboratorio de Investigación en Parasitología, Hospital Infantil de México Federico Gómez, México, D.
   F., México

12. IDENTIFICATION OF NOVEL REGULATORY MECHANISMS OF THE MULTIFUNCTIONAL 14-3-3
   PROTEIN IN GIARDIA DUODENALIS

                   a                            b                     b                                 a
   Marco Lalle , Anna Maria Salzano , Marco Crescenzi and Edoardo Pozio
   a                                                                                              b
    Department of Infectious, Parasitic and Immunomediated Diseases, Department of Environment and
   Primary Prevention, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161 Rome, Italy. E-mail:
   marco.lalle@iss.it


13. IDENTIFICATION               AND       LOCALIZATION       OF     THE              CYSTEINE        PROTEINASE                     TvCP12   OF
   TRICHOMONAS VAGINALIS USING POLYCLONAL ANTIBODIES AGAINST A SYNTHETIC
   PEPTIDE FROM THE MOST DIVERGENT REGION OF THIS PROTEIN
   León-Sicairos, C. R. and Arroyo, R.
   Departamento de Patología Experimental. Centro de Investigación y de Estudios Avanzados del IPN
   (CINVESTAV-IPN). Av. I.P.N. # 2508, Col. San Pedro Zacatenco. CP 07360, México. D.F., México.
   Email: claudia_leonsicairos@yahoo.com.mx; rarroyo@cinvestav.mx


14. INFLUENCE OF TRICHINELLA SPIRALIS-INDUCED INTESTINAL INFLAMMATION ON A GIARDIA
   LAMBLIA INFECTION IN THE MURINE HOST
                 1                    1                    1             2                       1
   Norbert Müller , Nicole von Allmen , Selina Christen , Ursula Forster ; Bruno Gottstein , and Monika
           2
   Welle
                            1                          2
   Institutes of Parasitology and Veterinary Pathology , University of Berne, Berne, Switzerland


15. CRYPTOSPORIDIOSIS CIRCULATION IN DIFFERENT REPRESENTATIVE SAMPLES FROM
   MAPUTO PROVINCE, MOZAMBIQUE
   Emilia Noormahomed*, Noémia Nhancupe*, Catherine Kauffmann-Lacroix**, Carmen Mascaró***,
   Mauro M. Colombo****
   * Parasitology Sector, Department of Microbiology, Faculty of Medicine, Eduardo Mondlane University,
   PO Box 257 Maputo- Mozambique, e-mail: eraul@teledata.mz ** Laboratoire de Parasitologie et
                                                                             ***
   Mycologie Médicale, Centre Hospitalo-Universitaire de Poitiers, France          Departament of Parasitology,
   Institute of Biotechnology, University of Granada, Spain ****Dept. of Cellular Developmental Biology,
   University of Rome, La Sapienza, Italy


16. ANALYSIS    BY    RANDOM       AMPLIFIED        POLYMORPHIC     DNA        OF       CLOSELY      RELATED
   TRICHOMONAS VAGINALIS STRAINS
   Felipe Padilla-Vaca, Ana Estela Gamiño-Arroyo, Minerva Paola Barrios-Ceballos and Fernando
   Anaya-Velázquez.
   Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
   Guanajuato, Gto., México.


17. INTERNAL SIGNAL-DEPENDENT TARGETING OF GIARDIA ISCS INTO TRICHOMONAD
   HYDROGENOSOMES
   Petr Rada, Pavel Dolezal, and Jan Tachezy
   Department of Parazitology, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic


18. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE EHRABB GENE PROMOTER
   OF ENTAMOEBA HISTOLYTICA
                        1                   2                  1                            1
   Mónica Romero-Díaz , Consuelo Gómez , Esther Orozco , and Mario A. Rodríguez .
   1
   Departamento de Patología Experimental. Centro de Investigación y de Estudios Avanzados del IPN.
                                                2
   A.P. 14-740, México, D.F. 07000, México. Programa Institucional de Biomedicina Molecular, ENMyH-
   IPN, Guillermo Massieu Helguera No. 239. Fracc. La Escalera. Ticomán, CP 07320, México, D.F.,
   México.
19. IDENTIFICATION AND LOCALIZATION OF TVLEGU-1, ONE OF THE LEGUMAIN-LIKE CYSTEINE
   PROTEINASES OF TRICHOMONAS VAGINALIS
   Salas-Garrido C G*, Ortega-López J **, Arroyo R *
   *Departamento de Patología Experimental **Departamento de Biotecnología y Bioingeniería, Centro
   de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN). Av. IPN #2508, Col. San Pedro
   Zacatenco, CP 07360. México, D.F. México. E-mail: gerardito@lycos.com; rarroyo@cinvestav.mx


20. THE SECOND INHIBITOR OF CYSTEINE PROTEASES FROM ENTAMOEBA HISTOLYTICA,
   AMOEBIASIN-2, MAY FUNCTION EXTRACELLULARLY.
   Mirela Šarić, Anke Harsman, Sabine Riekenberg, Henning Scholze
   University of Osnabrueck, Dept. Biology/Chemistry,


21. TRANSCRIPTONAL RESPONSE OF TRICHOMONAS VAGINALIS TO DIFFERENT IRON SOURCE
   BY cDNA MICROARRAY
   Jyh-wei, Shin and Petrus Tang
   Department of Parasitology, Core Laboratory of Microarray, College of Medicine, National Cheng
   Kung University, Tainan, Taiwan and Molecular Regulation & Bioinformatics Laboratory, College of
   Medicine. Chang Gung University, Taoyuan, Taiwan.


22. CLONING AN EXPRESSION OF A GENE FRAGMENT ENCODING THE FUNCTIONAL DOMAIN OF
   CP65 CYSTEINE PROTEINASE INVOLVED IN THE TRICHOMONAS VAGINALIS CELLULAR
   DAMAGE.
                                1                               2                              1
   Eduardo Solano-González, María Elizbeth Alvarez-Sánchez, Victor Hugo Rodríguez-Vargas, Leticia
                                      1                    2*
   Avila-González, Jaime Ortega-López and Rossana Arroyo
                                                  1                                        2
   Departamento de Biotecnología y Bioingeniería , Departamento de Patología Experimental , Centro
   de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro
   Zacatenco, C.P 07360, México D.F., México. E-mail: rarroyo@cinvestav.mx


23. CHARACTERIZATION OF THE 5S RIBOSOMAL RNA GENE IN TRICHOMONAS VAGINALIS:
   ANALYSIS PUTATIVE RNA POLYMERASE III PROMOTER ELEMENTS.
                            1                    1                  2                          1
   Ana Lilia Torres-Machorro , Roberto Hernández , Joaquín Sánchez , Imelda López-Villaseñor
   1
   Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas,
   Universidad Nacional Autónoma de México. Apartado Postal 70-228, C.P. 04510, México D.F.,
           2
   México. Facultad de Medicina, Universidad Autónoma del Estado de Morelos. Avenida Universidad
   1001, Cuernavaca Morelos, C.P. 62210, México


24. STRUCTURAL CHARACTERIZATION AND LOCALIZATION OF ANNEXIN E1 IN TROPHOZOITES
   OF GIARDIA LAMBLIA
   Anke Vahrmann, Dennis Priess, Anna Szkodowska, Tilly Bakker-Grunwald, and Henning Scholze
   Department of Biology/Chemistry, Biochemistry, University of Osnabrueck, Germany
25. IDENTIFICATION OF TRICHOMONADS IN HUMAN TISSUES BY MOLECULAR TOOLS
                             1                   2                             3             3
   Christophe DUBOUCHER , Stéphanie CABY , Nausicaa GANTOIS , Magalie CHABE , Isabelle
                    3                 2                                    4                 2
   DURAND-JOLY , Christophe NOËL , Pilar-DELGADO-VISCOGLIOSI , Monique CAPRON , Eduardo
           3                 5                        2
   DEI-CAS , Gerard MOREL , and Eric VISCOGLIOSI
   1                                                                   2
   Laboratory of Pathology, Saint-Germain-en-Laye Hospital, France; Inserm U547, Institut Pasteur of
                3                                                                       4
   Lille, France; Laboratory of Ecology and Parasitism, Institut Pasteur of Lille, France; Department of
                                                                5
   Water and Environment, Institut Pasteur of Lille, France; CNRS UMR 5123, University Claude
   Bernard, Lyon, France.
                 Sunday 18th September 2005

                          9:00-12:35

                     CELL BIOLOGY
Chairpersons: Johannes Hackstein and Marcos Vannier dos Santos
MYOSIN IB AND CYTOSKELETON ACTIVITIES DURING PHAGOCYTOSIS IN ENTAMOEBA
HISTOLYTICA

Sabrina Marion and Nancy Guillén
Cell Biology of Parasitism Unit, INSERM U389, Institut Pasteur, France nguillen@pasteur.fr

Phagocytosis of human cells by the pathogenic protozoan Entamoeba histolytica is a hallmark of
amoebiasis. This infectious disease, leading to intestinal dysentery and formation of liver abscesses, is
characterized by an extensive destruction of human tissues caused by killing and phagocytosis of human
cells. The complex process of phagocytosis can be dissected in several steps that have been characterized
according to morphological and biochemical criteria: (i) phagocytosis is initiated by the binding of particles or
cells to surface receptors of the phagocytic cell; (ii) the interaction between ligands and receptors triggers a
series of events, including reorganization of the cytoskeleton that leads to pseudopod extension; (iii) the
pseudopods close around the particle to form a phagosome, which fuses sequentially with the early and late
endosomes; (iv) finally the phagosome fuses with lysosomes, leading to digestion of the internalized
particles. We used magnetic beads covered with proteins from human serum as a model system to study the
early events involved in phagocytosis by E. histolytica. We validated the system showing that the beads
uptake triggered the activation of the actin-myosin cytoskeleton and involved a PI3-Kinase as previously
described for erythrophagocytosis. We purified early phagosomes from wild type amoeba and from parasites
that overproduced myosin IB (MyoIB+), the unique unconventional myosin of E. histolytica. The MyoIB+ cells
exhibit a slower and more synchronized uptake process than the WT strain. Proteomic analysis by LC/MS-
MS of the WT and MyoIB+ phagosomes allowed us to identify for the first time, molecular actors involved in
the early step of the uptake process. These include proteins involved in cytoskeleton activity, signaling,
endocytosis, lytic activity and cell surface proteins. Interestingly, the proteins that we found specifically
recruited on the phagosomes from the MyoIB+ strain, were previously described in other eukarytotic cells, as
involved in the regulation of cortical F-actin dynamics, such as α-actinin and formins. A protein homologuous
to D. discoideum Carmil, which binds myosin IB, was recruited to early phagosomes.This proteomics
approach allows a step further towards the understanding of the molecular mechanisms involved in
phagocytosis in E. histolytica that revealed some interesting differences compared to phagocytosis in
macrophages or Dictyostelium discoideum, and allowed to identify putative candidates for proteins linked to
myosin IB activity during the phagocytic process.
FURTHER STUDIES IN PHAGOCYTIC ACTIVITY OF TRICHOMONAS VAGINALIS

Antonio Pereira-Neves and Marlene Benchimol
Laboratório de Ultraestrutura Celular, Universidade Santa Úrsula, Rua Jornalista Orlando Dantas, 59,
Botafogo, Rio de Janeiro, Brazil

INTRODUCTION. The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomonosis,
the most common non-viral sexually transmitted disease in humans. It is understood that T. vaginalis has a
high phagocytic capacity once phagocytosis of various cells types by the parasite has been described both in
vivo and in vitro. However, we do not know if T. vaginalis is a naturally active phagocytic cell or if it is a cell
able to become active through cell signalizing by any specific chemicals, as occurs in macrophages.
MATERIALS AND METHODS. In attempt to make clear the question mentioned above, we cultivated Jt
strain of T. vaginalis in TYM Diamond’s medium for 24 h at 37ºC under follows conditions: in medium without
and with LPS (Lipopolysaccharides) 100 ng/mL. LPS are a major constituent of the cell wall of Gram-
negative bacteria and they are used in vitro, in a routine way, to activate macrophages (Adams et al, 1990;
Cunha et al, 1993; DaMatta et al 2000; Liew, 1991). Phagocytosis assays were made to analyze the
phagocytic rates of both cultures. In these assays, both cultures of T. vaginalis, after 24 h of growth, were
incubated in TYM medium for 60 min at 37ºC with yeast cells at a 1:100 parasite:yeast cells ratio. After
interaction, samples were processed for transmission and scanning electron microscopy. RESULTS AND
CONCLUSIONS. Similar results were obtained in both cultures of T. vaginalis. The parasite was able to
phagocytose the yeast cells. We not found a significantly expressive phagocytic rate between the parasites
grown in medium with LPS and those parasites grown in medium without LPS. We observed that the
parasites changed to an amoeboid morphology during the internalization of yeast cells. We noticed that T.
vaginalis are actively phagocytic over the whole cell surface by a mechanism similar to that described for
professional phagocytes, where pseudopodia are extended toward the target cell, which is subsequently
engulfed. During the internalization of yeast cells, parasites in different stages of mitoses were frequently
seen. Thus, our results suggest that T. vaginalis might be a natural phagocytic cell or that it might be not
stimulated by LPS.
ESV ORGANIZATION AND MATURATION IN GIARDIA LAMBLIA
            1            1          2              2
A. B. Hehl , S. Stefanic , D. Palm , S. G. Svard
1                                                             2
    Institute of Parasitology, University of Zürich, Switzerland; Department of Cell and Molecular Biology,
Uppsala University, Sweden

Formation of an environmentally resistant cyst wall – the synthesis and secretion of an extracellular matrix of
at least three cyst wall proteins (CWPs) and GalNAc homopolymers – in the protozoan parasite Giardia
lamblia is essential for its transmission. The cyst wall material is exported from the ER and accumulates in
developing encystation-specific vesicles (ESVs). The large, spherical ESVs are the only identifiable Golgi-
like cisternae in Giardia and represent a unique form of Golgi neogenesis. ESVs and their cargo undergo
maturation processes, which involve retrograde transport by COPI coated vesicles and presumably
retrotranslocation and proteasome-dependent degradation. The latter is reminiscent of protein quality control
mechanisms observed in the ER of higher eukaryotes. Specifically, we will discuss recent results showing
that proteasome complexes are recruited from peripheral sites in the cell to ESV membranes during the first
phase of encystation. Our data indicate that the 26S proteasomes are associated with the cytoplasmic face
of the ESV membrane, suggesting massive retrotranslocation of cargo proteins from these compartments.
Current work is focussed on the identification of additional components of this machinery and their functional
characterization.
APOPTOSIS IN GIARDIA AND OTHER ANAEROBIC PROTOZOANS

Naomi Kitchener ., Kate Topping and Tim Paget
Department of Biological Sciences, Hardy Building, The University of Hull, Cottingham Road, Hull. HU6
7RX. UK

The death of cells by apoptosis is a vital mechanism for maintaining developmental and homeostatic
processes within multicellular eukaryotes. Mitochondrial regulation of caspase activation results in
characteristic biochemical and morphological changes which were originally considered a multicellular
eukaryotic trait.
Over the past ten years, research has unearthed evidence of PCD mechanisms within increasingly “simple”
unicellular organisms. This study shows the existence of a form of PCD within the amitochondrial unicellular
protozoan parasite G. intestinalis.
Biochemical, flow cytommetry, light and fluorescent microscopy methods were used to investigate the effects
of pro-apoptotic drugs on the organism. The drugs were able to induce a form of cell death which exhibited
key biochemical and morphological indicators of apoptosis. The externalisation of phosphatidylserine and
DNA fragmentation were observed within the cells and were shown to be dose responsive.
This evidence along with that from studies on a number of other “anaerobic” protozoan suggest that PCD
has existed throughout eukaryotic evolution and has evolved to demonstrate incredibly diverse mechanisms
within different eukaryotic species.
CELL DIVISION AND FLAGELLAR DEVELOPMENTAL CYCLE OF GIARDIA
                1                2                    3
Eva Nohynkova , Pavla Tumova and Jaroslav Kulda
1
National Reference Laboratory for Diagnostics of Tropical Parasitoses, Faculty Hospital Bulovka,
                                     2                                 st                          3
Studnickova 7, 128 00 Prague 2, Department of Tropical Medicine, 1 Faculty of Medicine and Department
of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2, Czech
Republic.

Giardia is a bi-nucleated diplomonad equipped with four pairs of flagella of distinct location and function. The
flagella and fibrillar appendages of their basal bodies are grouped in two separated mastigonts that are
arranged in an incomplete axial bi-radial symmetry. The flagellar apparatus of Giardia undergoes profound
but poorly understood reorganization during cell division that is necessary to maintain heterogeneity of
flagella in progeny. We examined this process by using light and electron microscopy and
immunofluorescence allowing discrimination between the parent and newly assembled flagella. Results: (1)
In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops
separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the
early phase of mitosis. (2) We demonstrated the presence of a flagellar developmental cycle in Giardia.
During the cycle flagella of a dividing cell migrate, assume different position and transform to different
flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes
three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a
privileged basal body at which the microtubules of the adhesive disc nucleate. In principle the flagellar
maturation process in Giardia conforms to maturation of basal bodies observed in some unicellular algae as
well as to maturation of centrioles in animal cells. Demonstration of the flagellar cycle in a metamonad protist
Giardia suggests that the basal body/centriole maturation is a universal, evolutionary conserved
phenomenon representing one of core attributes of a eukaryotic cell.
RECENT ADVANCES IN BLASTOCYSTIS RESEARCH – IMPLICATIONS FOR PROTOZOAN
PROGRAMMED CELL DEATH AND PARASITE SURVIVAL

Kevin SW TAN, Manoj K PUTHIA, AMA NASIRUDEEN, Geok Choo NG
Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, National University of
Singapore, 5 Science Drive 2, Singapore 117597

Blastocystis hominis is an intestinal protozoan with unusual biological features. We will highlight recent work
done in our laboratory that provides new information on the complexity of cell death pathways and the
presence of Immunoglobulin A proteases in Blastocystis.


1. Programmed cell death (PCD) in Blastocystis - We demonstrated previously that a cytotoxic monoclonal
    antibody (MAb) 1D5 elicits a PCD response in Blastocystis hominis and showed that caspase-3-like
    protease influences but is not essential for PCD in MAb 1D5-treated B. hominis. We also showed that
    mitochondrial dysregulation played a role in cell death. We further analyzed the signaling pathways
    involved in PCD mediated by MAb 1D5. Blastocystis hominis cells were treated with MAb 1D5 or control
    MAb 5, either with or without pretreatment with a pan-caspase inhibitor, zVAD.fmk, and/or a mitochondrial
    transition pore blocker, cyclosporine A (CA). Flow cytometric examination of cell size, mitochondrial
    membrane potential (ΔΨm), caspase activation and in situ DNA fragmentation showed that zVAD.fmk and
    CA, used independently or in combination, failed to inhibit MAb 1D5-mediated PCD. Interestingly, cell
    exposure to either inhibitor resulted in partial inhibition of DNA fragmentation while combined exposure of
    cells to inhibitors abolished DNA fragmentation completely. This study sheds new light on the conserved
    nature of PCD pathways in parasitic protozoa and is also the first report describing caspase- and
    mitochondria-independent cell death pathways in a protozoan parasite.


2. Immunoglobulin A (IgA) proteases of Blastocystis - Microbial IgA proteases cleave human secretory IgA,
    promoting the mucosal adhesion of pathogens. To investigate if the enteric protozoan Blastocystis
    degrades human secretory IgA, cell lysate and conditioned medium from two species were exposed to
    immunoglobulin A. Secretory IgA was cleaved by both cell lysate and conditioned medium with mainly
    cysteine proteinase activity in B. hominis B isolate and aspartic proteinase activity in B. ratii WR1 isolate.
    These findings suggest that Blastocystis proteases may play a role in parasite survival in vivo.
"ENDOMICROBIA": CYTOPLASMIC SYMBIONTS OF TERMITE GUT PROTOZOA FORM A
SEPARATE PHYLUM OF PROKARYOTES

Andreas Brune
Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

In the hindgut of wood-feeding lower termites, lignocellulose is digested with the aid of a symbiotic
microbiota comprising both prokaryotes and protozoa. Although recent investigations have uncovered a vast
phylogenetic diversity among the gut symbionts, only little is known about the metabolic functions of the
individual populations and their interactions with each other and with the host. The largest fraction of the gut
microbiota consists of oxygen-sensitive flagellates affiliated with the Parabasalia or Oxymonadida. Based on
a few studies of parabasalid flagellates, it had been tacitly assumed that all termite gut protozoa ferment
cellulose to acetate, CO2, and H2. However, microinjection of radiolabeled metabolites into intact hindguts of
Reticulitermes species indicated that a considerable portion of the carbon flux proceeds via lactate. Also the
absence of hydrogenosomes in oxymonad flagellates and the significant impact of oxygen on carbon and
electron flow require the metabolism of the gut protozoa and their associated bacteria to be reconsidered.
Especially the larger flagellates of Reticulitermes species harbor numerous prokaryotic endosymbionts of so-
far unknown identity and function. Using a 16S rRNA-based, full-circle molecular approach, we showed that
the endosymbionts of Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) are
specific for their respective host flagellate and fall into the so-called Termite Group 1, a group of clones
previously obtained exclusively from gut homogenates of Reticulitermes speratus. The endosymbionts were
described as members of the candidate genus "Endomicrobium". An extended survey of other termite
species revealed that related lineages are present in and also restricted to the guts of all lower termites and
wood-feeding cockroaches of the genus Cryptocercus – a distribution pattern that coincides exactly with that
of the gut flagellates. They are phylogenetically quite diverse and only distantly related to other bacteria.
Therefore, we proposed the candidate phylum "Endomicrobia" for these unique bacteria with an
endosymbiotic lifestyle and of so-far unknown metabolic function.
COMMON MODE OF PROTEIN TARGETING INTO MITOSOMES AND HYDROGENOSOMES
                1                1           1                     1                 2                1
Pavel Dolezal , Ondrej Smid , Petr Rada , Zuzana Zubacova , Dejan Bursac , Robert Sutak , Jan
            1                2               1
Nebesarova , Trevor Lithgow & Jan Tachezy
1
Department of Parasitology, Charles University in Prague, Vinicna 7, 128 44 Prague, Czech Republic
2
Molecular Science and Biotechnology Institute, University of Melbourne, Parkville 3010, Australia

Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. However, some
unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from
the eukaryotic lineage before the acquisition of the pre-mitochondrial endosymbiont. Recently, their
amitochondrial status was challenged by the discovery of double membrane-bound organelles named
mitosomes, which may represent highly reduced mitochondria. Here we report that proteins involved in FeS
cluster assembly (IscS, IscU and [4Fe4S]-ferredoxin) are targeted into mitosomes of Giardia intestinalis by
means of N-terminal or internal targeting signals, which are recognized by the mitosomal import machinery.
When the mitosomal proteins were expressed in Trichomonas vaginalis, they were specifically delivered into
hydrogenosomes, a hydrogen-producing form of mitochondria, and processed by a metalloprotease. This
shared mode of protein targeting strengthens the hypothesis that mitosomes, hydrogenosomes and
mitochondria represent different forms of the same fundamental organelle that have evolved under distinct
selection pressures.
THE CENTROSOMES AS A GIARDIA LAMBLIA CELLULAR CONTROL CENTER IN
REGULATING DIFFERENTIATION
                    1             1               5                        2                      2              3
Tineke Lauwaet , David Reiner , Edwin Romijn , Shanda R. Birkeland , Michael J. Cipriano , Daniel Palm ,
                        1                4                  3                       2                 5
Barbara J. Davids , Sarah E. Pacocha , Staffan G. Svard , Andrew G. McArthur , John Yates III , Frances D.
         1
Gillin
1
    Department of Pathology, University of California San Diego, CA 92103;
2
    Marine Biological Laboratory, Woods Hole, MA 20543
3
    Department of Cell and Molecular Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala,
Sweden
4
    Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA 02543
5
    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037

             Giardia lamblia is of both medical importance and of fundamental biological interest. Giardial
genome and SAGE transcriptome analyses have advanced greatly. Because we can complete its life cycle
in vitro, this protozoan is an excellent model for study of regulation of growth vs differentiation. The ability of
the complex giardial cytoskeleton to undergo dramatic remodeling throughout the life cycle in response to
rapidly changing signals from the host and the external environment is central to its success as a pathogen.
We found earlier that the protein kinase A (PKA)-cAMP and calcium signaling pathways regulate different
steps in the resumption of motility and cytokinesis during the cellular awakening of excystation. Recently, we
showed that protein phosphatase PP2A helps regulate the trophozoite’s decision to cease growth and
encyst. Moreover, PKA, calmodulin, and PP2A all co-localize with centrin to the flagellar basal bodies or
centrosomes. Importantly, PKA and PP2A also localize to other, cytoskeletal structures that are unique to
Giardia. PP2A localizes to the ventral or attachment disc. PKA and PP2A target to different paraflagellar
dense rods. The functions of the dense rods are not known, but the targeting of PKA and PP2A to these
structures decreases in response to specific signals from the host.
             We propose that the centrosomes may be a cellular “control center” while other cytoskeletal
elements may have specialized effector functions that are reflected in their distinct protein compositions.
Therefore, we isolated centrosomes from Giardia cytoskeletons with sucrose density gradients. Fractions
were analyzed by IFA, ELISA and Coomassie stained 1D protein gels for the presence of PKA, PP2A and
centrin. The centrosome rich fraction was analyzed by Multidimensional protein identification technology
(MudPIT) which revealed 174 putative centrosomal proteins of which ~ half are unique to Giardia or
“hypothetical”. Moreover, SAGE analysis demonstrated a subset of ~24 orfs that is highly upregulated in
cysts and early excystation. In support of our hypothesis, epitope tagging shows that all six of the “new”
proteins tested so far, localize to the centrosomes and most also target to other cytoskeletal elements.
       These studies will give us a unique picture of the distinct functions of key elements of the cytoskeleton
in mediating signaling responses to physiologic stimuli throughout the giardial life cycle.
ASSEMBLY AND DISASSEMBLY OF THE ADHESIVE DISC DURING CELL DIVISION OF A
WILDTYPE AND ALBENDAZOLE TREATED GIARDIA INTESTINALIS
                1                2                    3
Eva Nohynkova , Pavla Tumova and Jaroslav Kulda
1
National Reference Laboratory for Diagnostics of Tropical Parasitoses, Faculty Hospital Bulovka,
                                        2                                        st
Studnickova 7, 128 00 Prague 2,          Department of Tropical Medicine, 1           Faculty of Medicine, and
3
Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 44 Prague 2,
Czech Republic.

Trophozoites of Giardia are equipped with a special organelle of attachment, the ventral adhesive disc. This
organelle, essential for survival and pathogenicity of the parasite in the host intestine is considered to be the
main target for albendazole, a benzimidazole drug with anti-microtubular activity. With aid of light and
electron microscopy and immunofluorescence staining we followed structural changes accompanying the
assembly and disassembly of the adhesive disc during cell division of Giardia intestinalis in culture and effect
of albendazole on these processes. We found that dividing Giardia alternates attached and free swimming
phases in accordance with a particular phase of the division and adhesive competence of the parent or
newly assembled daughter discs. At early mitosis disc microtubules detached from basal bodies thus
enabling migration and reorganization of flagella. Orderly disassembly of the parent disc skeleton followed,
relative to a gradual shortening and eventual loss of the giardin microribbons. Collapse of the disc chamber
and retraction of the ventrolateral flange resulted in parasite detachment. The adhesive discs of daughter
cells begun to assemble during the attached phase on the anterior dorsal side of the parent cells and
completed their development during the swimming phase before the progeny splitting was terminated. The
daughter cells still connected tail to tail by a cytoplasmic bridge attached to a substrate by their fully
assembled discs and terminated the division by a process resembling adhesion dependent cytokinesis of
amoeboid cells. Our observation on albendazole treated Giardia showed that the drug effect is directly
associated with the cell division. Albendazole affected only those cells that entered the M phase of cell cycle.
The drug showed selective effect on microtubules of the mitotic spindle and the adhesive disc. Consequently
the mitosis ceased and assembly of the daughter adhesive discs was inhibited. Also disassembly of the
parent disc showed abnormalities. Although the mitosis was stopped at early phase, the cytokinesis
continued with undisturbed pattern of distribution and de novo synthesis of flagella. Resulting octoflagellated
progeny lacked adhesive disc and possessed one, two or no nuclei. These results indicate, that a functional
spindle assembly checkpoint is absent in Giardia.
         Sunday 18th September 2005

                14:30 – 18:05
            Molecular biology
Chairpersons: Patricia Johnson and Jan Tachezy
THE TRICHOMONAS VAGINALIS GENOME SEQUENCING PROJECT

Joana C. Silva
The Institute for Genomic Research, Rockville MD (USA)

The sequencing and automated annotation phases of the Trichomonas vaginalis Genome Project are now
completed, and extensive analyses of the structure and composition of the genome are underway. The
genome is estimated to be approximately 177 Mb long, and has been found to be extremely repetitive.
Independent estimates suggest that 40% of the genome consists of highly repeated sequences. This
characteristic has had a considerable effect on the assembly, which is currently composed of approximately
17,000 scaffolds. Analyses of the repetitive component of the genome revealed that a few dozen repeats,
each represented on average by hundreds of copies, account for most of its bulk. These repeats consist of
several transposable elements, such as Tvmar1, the first mariner element described from a protist, and also
virus-like particles, such as a Pox-D5-like repeat, and T. vaginalis gene families, such as ABC transporters.
Our analyses show that the level of polymorphism within each repeat family is remarkably low, and that the
expansion of most repeat families post-dates the split of T. vaginalis from its sister taxon, T. tenax. These
results will be discussed in the context of the biology of T. vaginalis.
GENOTYPING TRICHOMONAS VAGINALIS
                1                1                      2
Peter Upcroft , Jacqui Upcroft and Patricia Johnson
1
Queensland Institute of Medical Research, 300 Herston Rd, Brisbane, Queensland 4006, Australia.
2
    University of California Los Angeles, California 90095-1489, USA.

          We have used pulsed-field gel electrophoresis (PFGE) as an analytical tool to separate large DNA
molecules of Trichomonas vaginalis for genome fingerprinting as routinely used to monitor bacterial
infections. Although high molecular weight T. vaginalis DNA has been extracted and genes cloned, no
consistent electrophoretic karyotype has yet been described because of apparent DNA degradation (due to
the highly active endogenous nucleases) and the large size of the genome.
          High molecular weight DNA was prepared from Trichomonas vaginalis laboratory isolates from
Australia and USA, clinical isolates from South Africa, Papua New Guinea and other parts of the world. The
DNA was macrorestricted with the enzyme SmaI such that 50 kb was the minimum size segment. Segments
were separated by pulsed field gel electrophoresis and hybridised with a variety of gene probes. Each isolate
was distinguished by each of the probes and each isolate was unique. Four single copy gene probes (fd,
hmp35, ibp and pfoD) were identified but probes which identified several bands (pfoB and α-scs) per isolate
were most informative for genotyping. The hydrogenase gene probe hybridised with many bands indicating
multiple copies or cross-hybridising genes in the genome and was not a useful genotyping probe. The
pyruvate:ferredoxin oxidoreductase (PFO) B gene probe was most informative with 3 to 7 copies per
genome and with unique banding patterns for each isolate. When the PFOB probe was used with smaller
restriction fragments (mean size around 50 kb using the restriction enzyme XbaI for example) differences
between isolates were not always obvious. This indicates that gene proximal regions are more conserved
than distal regions.
          Up to 50% of isolates, depending on the source, carry a clearly discernable Mycoplasma hominis
genome each of which is as diverse and identifiable as the genome of the host trichomonad.
          We propose that genes such as the PFOB gene probe in conjunction with SmaI cleaved
Trichomonas DNA can now be used to identify epidemiological linkage between infections, to follow drug-
resistant isolates and determine whether particular strains are associated with highly symptomatic infections.
          This recent development of a genotyping and genomic mapping method coupled with in situ
hybridisation provides a backbone on which to place genome sequence data from the TIGR/UCLA Genome
Sequencing Project. For example, we have identified 96 candidate ABC transporters, 42 MDR-like genes,
and 77 oxidoreductases, all of which may be involved in clinical drug resistance. These data will be
invaluable for defining new drug targets and the potential of novel drugs that we have developed.
INSIGHTS FROM THE TRANSCRIPTOME OF TRICHOMONAS VAGINALIS

Petrus Tang
Molecular Regulation & Bioinformatics Laboratory, College of Medicine. Chang Gung University, Taoyuan,
Taiwan.

The Trichomonas vaginalis Expressed Sequence Tag (TvEST) Project have generated more than 60,000
ESTs from seven representative cDNA libraries related to the cell division cycle, growth and pathogenesis of
the parasite for the study of Trichomonas comparative transcriptomics (http://cgbc.cgu.edu.tw/est/).
Clustering of the ESTs revealed that the Trichomonas transcriptome contain at least 15,000 uniqenes, the
largest gene repertoire identified in parasitic protozoa so far. Among them, only 35% of the unigenes can be
assigned a putative function based on significant sequence homology to know proteins or protein
motifs/domains. Around 90% of the identified unigenes can be mapped to the Trichomonas A1 genome
being under sequencing by The Institute of Genomics Research (TIGR). Although the percentage of
tentative clusters and singletons are 55% and 45% respectively, over 90% of the transcripts are expressed
at very low levels. Less then 2% of the unigenes are expressed in all the libraries. These genes may be
essential for the viability, growth and survival of the parasite. The number of unigenes specifically expressed
in the trophozoite, G2 phase, cold-induced pseudocyst, vaginal epithelail cells-mediated cytoadherence,
fibronectin-mediated cytoadherence, low iron culture and low iron culture cDNA libraries are 1678, 976,
1108, 326, 1539 and 725 respectively. The database generated from the present work allowed us to study
the global gene expression of T. vaginalis in a more comprehensive view. One of the many applications is to
define the kinase/phosphatase complement of the parasite (kinome/phosphatome) and to decipher the
complex network of phosphorylation-based signaling. More than 450 kinases are identified, but only around
150 phosphatase are present in the transcriptome. It implies that either most phosphates are only transiently
in place or the phosphorylation-dephosphorylation process is not always a one-to-one relationship. The near
completion of the T. vaginalis genome sequencing project and the differential gene expression patterns
elucidated by the EST project, together with the advances in Trichomonas microarray and 2-dimensional gel
electrophoresis technologies has made it possible to use T. vaginalis as a model organism to study the
“Systems Biology” of protozoa.
A NOVEL PROTEIN, Myb24, REGULATES TRANSCRIPTION OF THE IRON-INDUCIBLE ap65-
1 GENE INTHE PROTOZOAN PARASITE TRICHOMONAS VAGINALIS
                1                 1                2                 1                      2, †
Shiou-Jeng Ong , Hong-Ming Hsu , Hsing-Wei Liu , Chien-Hsin Chu and Jung-Hsiang Tai
1                                                                                   2
Department of Parasitology, College of Medicine, National Taiwan University, and Division of Infectious
Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.

        Transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with
changes in the iron supply or growth stages. In the present study, two Myb recognition elements, MRE-1/MRE-2r
and MRE-2f, were found to synergistically regulate growth-related activity, but antagonistically regulate iron-
inducible activity, of an episomal ap65-1 promoter. A myb24 gene, which was identified from Southwestern
screening of the MRE-2f-binding proteins, was found to express as a 0.7 kilo-base transcript with a non-
canonical start site. The Myb24 protein was detected as a major 35-kDa protein in nucleus and cytoplasm with
variable intensities upon changes of the iron supply. A small region rich in basic amino acid residues following
the R2R3 DNA binding domain was found to contain a nuclear localization signal. A recombinant Myb24 was
shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro, and this activity is regulated C- terminus
of Myb24. Overexpression of a tagged Myb24 in T. vaginalis resulted in depressed ap65-1 transcription at an
early growth stage, but enhanced this transcription at a later growth stage, with concomitantly diminished iron-
inducible ap65-1 transcription at both stages. The tagged Myb24 was found to constantly occupy the
chromosomal ap65-1 promoter at a proximal site, but differentially select two distal sites only at a later growth
stage. Together, these observations suggest that Myb24 critically regulates multifarious ap65-1 transcription,
possibly via differential selection of multiple promoter sites in response to environmental changes.
SPLICEOSOMAL INTRONS IN TRICHOMONAS VAGINALIS
                    1               2                 3                        2
Stepanka Vanacova , Weihong Yan , Jane M. Carlton and Patricia J. Johnson
1                                                                              2
Department of Cell Biology, Biozentrum, University of Basel, Switzerland, Department of Microbiology,
                                                                                           3
Immunology & Molecular Genetics, University of California, Los Angeles, USA and                The Institute for
Genomic Research, Rockville, Maryland, USA

Metazoa have evolved elaborate splicing mechanisms to remove introns that would otherwise disrupt the
capacity of genes to encode proteins. Splicing requires regulatory motifs in the intron and is mediated by a
ribonucleoprotein complex, the spliceosome. We demonstrate the presence of a splicing apparatus in the
deep branching protist Trichomonas vaginalis and show that regulatory motifs found in yeast and metazoa
introns are required for splicing. We also describe the first intron-containing genes in this lineage. These
introns share a highly conserved extended 3' splice site (SS) motif with the only described intron from a gene
in another deep-branching eukaryote, Giardia intestinalis. This motif, which also encompasses the branch
point, is necessary for splicing. These studies demonstrate the conservation of intron regulatory elements
across large evolutionary distances, reveal unexpected motif conservation in deep-branching lineages and
suggest that a simplified mechanism of splicing is employed by primitive unicellular eukaryotes. Moreover,
the position of introns in T. vaginalis genes are conserved in orthologous genes from yeast and metazoa
indicating their presence in the genes of a common ancestral eukaryote.
COMPREHENSIVE            ANALYSIS        OF     VESICULAR        TRAFFICKING          IN    ENTAMOEBA
HISTOLYTICA

Tomo Nozaki
Gunma University Graduate School of Medicine, Japan

Vesicular (membrane) trafficking plays an indispensable role in the parasitism and pathogenesis of the
protozoan parasite Entamoeba histolytica. Amebic trophozoites ingest and feed on microorganisms and
mammalian cells. The amebae also secrete a number of degradative molecules including cysteine proteases
and amoebapores responsible for the destruction of host tissues. Although recent completion of the
Entamoeba genome shed light on the extreme complexity of membrane trafficking in this parasites (e.g., >
90 Rab genes), molecules and molecular mechanisms involved in vesicular trafficking remain largely
unknown. In this presentation our on-going studies to attempt to dissect molecular mechanisms of vesicular
trafficking in this parasite will be presented. First, proteomic analysis of phagosomes revealed a panel of
>170 proteins that either compose phagosomes or are transported by phagosomes. We demonstrated
dynamic changes of phagosome proteins during phagosome maturation, as well as variations of phagosome
proteins between isolates. We also verified both presence and kinetics of representative phagosome
proteins by immunoblot, immunofluorescence, and video microscopy. Second, we have established video
microscopy to monitor vesicular pH, degradation of ingested materials, and the movement of GFP-tagged
amebic proteins (e.g., Rab or FYVE domain-containing proteins), in a living cell. We demonstrated, using the
system, the dynamism of phagosome acidification and degradation of its content. Acidification of
phagosomes occurred very rapidly (<1.5-2 min) and persisted for >12 h, while degradation of ingested
Leishmania promastigotes occurred within 20 min. We also observed remarkable differences in the kinetics
of phagosome maturation between E. histolytica and its related and non-pathogenic species E. dispar and
between highly virulent and avirulent strains of E. histolytica. Third, we have analyzed gene expression
profiles by DNA microarrays. We attempted to determine vesicular trafficking genes responsive for the
ameba virulence by comparing expression profiles of ameba lines showing different virulent competence.
These studies should provide molecular basis of pathogenesis of this important pathogen.
EPIGENETIC REGULATION OF GENE EXPRESSION IN THE PROTOZOAN PARASITE
ENTAMOEBA HISTOLYTICA.

Ankri Serge, Ohad Fisher, Hala Harony, Mona Abed, Sabina Bernes, Tal Lavi and Rama Siman-Tov.
Department of Molecular Microbiology, The Bruce Rappaport Faculty of Medicine, Technion, P.O.B 9649
Haifa 31096 Israel.

DNA methylation is an epigenetic modification that occurs in a wide range of prokaryotic and eukaryotic
organisms. DNA methylation is an important mechanism of regulation of genomic functions such as
differential control of gene expression. Our knowledge about the relevance of DNA methylation in the biology
of protozoan organisms is scanty. We identified by immunodetection the presence of 5-methylated cytosine
(m5C) in the parasite Entamoeba histolytica. The synthesis of m5C is catalyzed by a cytosine-5 DNA
methyltransferase (Ehmeth) that belongs to the Dnmt2 proteins family. The biological function of members of
the Dnmt2 family is unknown. To get insights into the cellular process(es) regulated by Ehmeth, we identified
by affinity chromatography analysis using anti-5mC antibodies as ligand, a number of methylated DNA
targets in the genome of E.histolytica. These DNA targets include LINE retrotransposon and heat shock
protein 100. Each of these targets has been studied separately and the effect of DNA methylation on their
expression will be discussed.
In addition, we altered Ehmeth levels by constitutive overexpression. Overexpression of Ehmeth resulted in a
pleiotropic phenotype including a lower growth rate, the accumulation of multinucleated giant trophozoites,
resistance to oxidative stress and decrease in cytopathic activity. These results indicate that Ehmeth is a key
regulatory element in controlling cellular processes in this protozoan parasite.
IDENTIFICATION IN GIARDIA LAMBLIA OF TWO EUKARYOTIC TRANSLATION INITIATION
FACTOR 4E HOMOLOGUES WITH DISTINCTIVE FUNCTIONS

Lei Li and C. C. Wang
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143
                                                                         7
         Eukaryotic translation initiation factor 4E (eIF4E) binds to the m GTP of capped mRNA and is an
essential component of the translational machinery that recruits the 40S small ribosomal subunit. Two eIF4E
                                                                             7
homologues were identified in the genome of Giardia lamblia. Using m GTP-Sepharose affinity column
chromatography, a specific binding protein was isolated and identified to be Giardia eIF4E2. The other
                                                  2,2,7
homologue, Giardia eIF4E1, binds only to the m        GpppN structure. Neither homologue can rescue the
function of yeast eIF4E. But a knockdown of eIF4E2 mRNA in Giardia by a virus-based anti-sense ribozyme
                                                 7
decreased translation, which was shown to use m GpppN-capped mRNA as template. Thus, eIF4E2 is likely
the cap-binding protein in the translation initiation complex. The same knockdown approach indicated that
eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of
both homologues in the cytoplasm. But eIF4E1 was found also concentrated and co-localized with the
 2,2,7
m    GpppN-cap, 16S-like ribosomal RNA and fibrillarin in the nucleolus-like structure in the nucleus.
eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein is bound to Giardia small nuclear
                                   2,2,7
RNA D and H known to have an m         GpppN-cap, thus suggesting a novel function not yet observed among
the other eIF4Es in eukaryotes.
MORE THAN 140 GENES HAVE BEEN ACQUIRED THROUGH HORIZONTAL GENE
TRANSFER FROM BACTERIA TO RUMEN CILIATES.
                 a                 b                          c                       a
Guénola Ricard , Neil R. McEwan , Johannes H.P. Hackstein and Martijn A. Huynen .
a
    Center for Molecular and Biomolecular Informatics, Nijmegen Center for Molecular Life Sciences, Radboud
University Nijmegen Medical Centre, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
b
    Rowett Research Institute, Aberdeen, AB21 9SB, Scotland
c
    Department of Evolutionary Microbiology, Radboud University Nijmegen, Nijmegen, The Netherlands

The horizontal transfer of genes into Ciliates from Bacteria which live in close proximity to them within the
rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). About 4000
ESTs were isolated from the two main types of rumen Cilates: Entodiniomorphs (e.g. Polyplastron
multivesiculatum, Epidinium ecaudatum, Eudiplodinium maggii, Entodinium caudatum, Entodinium simplex,
Diploplastron affine and Metadinium medium) and Vestibuliferida, previously called Holotrichs (e.g. Isotricha
prostoma, Isotricha intestinalis and Dasytricha ruminantium). A comparison of the sequences with the
completely sequenced genomes, followed by large-scale construction of phylogenies, identified 148 ciliate
genes that specifically cluster with genes from the Bacteria. Of these genes, 34 cluster with genes from the
Firmicutes, a phylum of Bacteria that is well represented in the rumen. This phylogenetic clustering, coupled
with the absence of close relatives of these genes in Tetrahymena, indicates that they have recently been
acquired via Horizontal Gene Transfer (HGT). Among these HGT candidates, we found an over-representation
(>70%) of genes involved in the anaerobic breakdown of complex carbohydrates, a rich food source in the
rumen. We propose that the acquisition of these genes has facilitated the Ciliates’ colonization of the rumen.
TEAMWORK: TRICHOMONAS VAGINALIS                       RNA POLYMERASE II AND THE INITIATOR
BINDING PROTEIN (IBP39)
                     1                1              1                1                        2
Patricia J. Johnson , Audrey O.T. Lau , Alias J. Smith , Mark T. Brown and Maria Schumacher
1
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles,
                 2
California, USA, Department of Biochemistry and Molecular Biology, Oregon Health & Science University,
Portland, Oregon, USA

The core promoter required for assembly of the transcription pre-initiation complex (PIC) in Trichomonas
vaginalis requires a highly conserved DNA initiator (Inr) element. The isolation and characterization of a T.
vaginalis Inr-binding protein (IBP39) demonstrated a 2 domain structure for this transcription factor: an N-
terminal Inr-binding domain (IBD) that is connected via a flexible linker to a C-terminal domain (C-domain).
Crystal structure analyses of the IBD revealed a novel winged-helix motif with prokaryotic and eukaryotic
features and a scaffold similar to that of ETS-family proteins. The IBP39 C-domain structure unveiled a
PYTS-specific binding pocket and biochemical studies indicate that this pocket interacts with the T. vaginalis
RNA polymerase II (RNAP II) large subunit carboxyl terminal domain (CTD). We present 2-hybrid,
immunoprecipation and kinetic analyses that demonstrate a direct interaction between the CTD and the C-
terminal domain of IBP39. Studies are underway to determine whether the phosphorylation state of the CTD
regulates the interaction of RNAPII and IBP39. Six homologues of the IBP39 gene have also been identified
in the T. vaginalis genome database, making gene knock-out experiments to test whether this protein is
essential tedious.
         Monday 19th September 2005

                 9:15 – 12:30
        Host-parasite relationships
Chairpersons: Michael Duchene and Nigel Yarlett
ENTAMOEBA HISTOLYTICA: EHADH112 IS A NOVEL MEMBER OF THE ALIX/AIP1 PROTEIN
FAMILY

Cecilia Bañuelos, Guillermina García-Rivera, Israel López, Leobardo Mendoza and Esther Orozco
Departamento de Patología Experimental, CINVESTAV IPN and Program of Genomic Sciences UACM,
Mexico, D.F.

The EhCPADH complex is a dimeric protein of Entamoeba histolytica, the protozoan responsible of human
amoebiasis. EhCPADH is formed by a cysteine protease (CP) and a protein with an epitope that is involved
in the trophozoites adherence to target cells (ADH). EhCP112 and EhADH112 are encoded by two adjacent
genes. By different strategies we have investigated the independent functions of each protein. Here we
report the functional characterization of EhADH112 protein. We found two homologous of Ehadh gene in the
parasite genome data bank. EhADH112 has the Bro1 domain at its amino terminus and a consensus context
for Src-tyrosine kinase phosphorilation, both involved in signal transduction. EhADH112 protein belongs to
the ALIX/AIP1 protein family and its functions might be associated with those found for these proteins in
other systems. By in silico analysis we found several ALIX/AIP1 interacting protein encoding sequences in
the E. histolytica genome databases. Some of these proteins are involved in endocytosis and a reduced
number of them have been cloned and studied by our group or for other groups, strongly suggesting the role
of EhADH112 protein in signal transduction during endocytosis. In the carboxy terminus the EhADH112
protein has the adherence domain. Trophozoites transfected with the Ehadh112 full length gene augmented
their adherence and phagocytosis efficiency. However, those transfected with the Bro1 domain exhibited
poor adherence to and phagocytosis of red blood cells. By confocal microscopy, we detected the Bro1
peptide forming multi-vesicular bodies in the cytoplasm, whereas the complete EhADH112 protein was
located also in the plasma membrane. Our results show that the EhADH112 has an adherence domain to
bind target cells and a Bro1 domain to perform signal transduction.
ENTAMOEBA HISTOLYTICA : EPIGENETIC SILENCING OF THE AMOEBAPORE GENE
RESULTS IN VIRULENCE - ATTENUATED STABLE TROPHOZOITES
                                                                               †
Rivka Bracha, Michael Anbar, Yael Nuchamowitz, Li Yan and David Mirelman
Department of Biological Chemistry, Weizman Institute of Science, Rehovot, Israel

Transfection of trophozoites of a virulent E. histolytica strain, HM-1:IMSS with a hybrid plasmid containing a
genomic copy of the amoebapore gene (Ehap-a), including its upstream and downstream regulating
elements, caused, instead of over-expression, a complete suppression of transcription of both, the episomal
and chromosomal gene [Bracha et al. (2003) Eukaryot. Cell, 2, 295-305].. Nuclear run-on experiments
revealed that gene silencing was at the transcription initiation level (TGS). Furthermore, removal of the
plasmid from silenced trophozoites and long term cultivation of cloned, plasmid-less trophozoites ( termed
G3 ) in the presence of inhibitors of DNA methyl transferases such as 5-Aza-Cytosine and Zebularine, or the
histone deacetylase inhibitors Trichostatin A or butyrate, failed to restore the expression of the Ehap-a gene.
Analysis of the 5’ upstream fragment ( 470 bp ) required for the triggering of gene silencing revealed that it
contained the promoter region of the Ehap-a gene, a T-rich stretch, followed by a truncated SINE (Short
Interspersed Element) that is transcribed from the antisense strand. Both, the T-rich stretch and sequences
of the 5’ SINE were essential for the transcription of the adjacent SINE element . RNA extracts from gene
silenced cultures showed small amounts of short (~140 n), single stranded RNA molecules with homology to
SINE1 but no siRNA. Chromatin immunoprecipitation (ChIP) analysis with an antibody against methylated
K4 of histone H3 revealed a de-methylation of K4 at the domain of the Ehap-a gene indicating transcriptional
inactivation.
The novel plasmid-less trophozoites (G3) were found to be non-virulent , they failed to kill mammalian
                                                                                              6
cultured cells and did not induce liver lesions in hamsters even at inoculations of 10            trophozoites
[Bujanover et al, Int. J. Parasitol. 33, 275-281,2003 ]. We have also demonstrated that in the absence of
amoebapore expression, the trophozoites didn’t cause amebic liver abscesses in the SCID mouse model .
Nevertheless, the amoebapore-less trophozoites still caused some inflammation and tissue damage in
infected human colonic xenografts [ Zhang et al. Infect. Immun. 2004. 72: 678-83 ] Preliminary studies using
intraperitoneal immunization of hamsters with the attenuated G3 strain showed that it evoked a significant
humoral response which partially protected hamsters from challenge by the virulent E. histolytica strain. The
virulence-attenuated trophozoites have the advantage of evoking an immune response to the same antigenic
epitopes as in the virulent strain, and our attempts to silence an additional virulence gene may lead to a
harmless, potential candidate for a live, oral vaccine against Amoebiasis.
A POSSIBLE FUNCTIONAL RELATIONSHIP BETWEEN SURFACE GLYCOPROTEINS OF
ENTAMOEBA HISTOLYTICA AND HUMAN PHAGOCYTES
              1               1               2                     1,3                       1
Marina Binder , Helen Melzer , Nancy Guillén , Alexandra Marinets , Gerhard Wiedermann , and Michael
          1
Duchêne
1
Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology,
                                    2
Medical University Vienna, Austria; Unit of Cellular Biology of Parasitism, Pasteur Institute, Paris, France;
3
currently living in Vancouver, Canada.

Entamoeba histolytica proteophosphoglycans (PPGs), previously called lipophosphoglycans (LPGs) or
lipopeptidophosphoglycans (LPPGs) are abundant glycoproteins on the surface of the parasite and within
vesicles. The monoclonal antibody EH5, raised in our laboratory, binds to these antigens and significantly
protected SCID mice against amoebic liver abscess. To investigate the EH5 epitope we had identified a
number of peptide mimotopes from phage display libraries which revealed a single consensus motif Gly-Thr-
His-Pro-X-Leu. We were able to identify a genomic sequence in the data from the E. histolytica genome
project which encoded a 52 kDa protein including exactly this consensus sequence. As would be predicted
from the carbohydrate structure the protein contained 60 serine residues which could be O-glycosylated via
phosphodiester bonds. A mouse serum raised against a synthetic peptide from another part of the coding
sequence gave the same binding pattern in western blots as the antibody EH5, supporting the view that we
had cloned the correct gene. So far, low levels of the recombinant protein were obtained, but these gave
strong western blot signals with the EH5 antibody. In another experiment the six mimotope consensus amino
acid residues were genetically fused into PH domains by themselves or with 6 or 12 flanking residues. All the
peptides displayed in an unrelated protein also bound to antibody EH5 confirming that the epitope was a
sequential protein epitope.
Surprisingly the EH5 antigen open reading frame was significantly similar to the CD68 antigen, also called
macrosialin, which is expressed by phagocytic cells of the immune system such as monocytes,
macrophages, granulocytes, and dendritic cells. Both in phagocytes and in amoebae, the antigen localizes to
the surface and inside vesicles. The EH5 antigen was found in vesicles with and without protease activity.
The antibody EH5 inhibited erythrophagocytosis in the amoebae, and currently we are investigating whether
the EH5 antigen may also play a role in the phagocytosis of bacteria.


The study was supported by grants BIO4-CT98-0121 from the European Commission and grant P15960
from the Austrian Science Fund.
THE FUNCTIONAL INTERFACE BETWEEN TRICHOMONAS VAGINALIS AND ITS HOST
CELL

John F. Alderete, Ashwini S. Kucknoor, Vasanth Mundodi, Ana Garcia and Te-Hung Chang
Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USA

Elucidation and characterization of virulence factors is necessary for understanding mechanisms of
pathogenesis. Infection of women by T. vaginalis occurs in the challenging urogenital environment that is
constantly changing, nutrient limiting, and undergoing immune surveillance. A brief summary of the
properties that contribute to successful colonization and host parasitism of Trichomonas vaginalis will be
presented, including the 1) association with host proteins, 2) evasion of the innate immune system, 3)
modulation of expression of genes by environmental cues, and 4) molecules and mechanisms of
cytoadherence. Recent work on successful gene silencing strategies and heterologous expression of T.
vaginalis genes in the bovine trichomonad affirm the functional diversity of enzymes as surface proteins
involved in vaginal epithelial cell (VEC) tropism. A previously unknown link between polyamine metabolism
and secretion of putrescine with masking of adhesins for modulating adherence to and cytotoxicity of VEC
has being elucidated. Novel surface protein immunogens of unknown function of T. vaginalis have been
identified, and characterization reveals sequence identity to transcription regulatory elements and metabolic
enzymes. In addition, T. vaginalis adherence mediates differential gene expression in VECs and increased
expression of parasite genes. A family of genes encoding trichomonad surface proteins, including adhesins,
α-actinin and a novel protein, have been found to be regulated in expression by independent signal
pathways involving iron and contact with VECs. Finally, the accumulated knowledge base on studies of
virulence properties and the biology of the host-T. vaginalis interface has resulted in a sensitive and specific
point-of-care antigen-based lateral flow diagnostic for women. Equally noteworthy is a second generation,
unprocessed urine test for T. vaginalis useful for diagnosis of infected men and women.
THE ROLE OF CELL SURFACE GLYCOCONJUGATES IN THE PATHOGENESIS OF
TRICHOMONAS VAGINALIS

Patrícia J.Johnson, Cheryl Y. Okumura, Felix D. Bastida-Corcuera and Linda G. Baum, Department of
Microbiology, Immunology and Molecular Genetics and Department of Pathology, University of California
Los Angeles, Los Angeles, CA.

Trichomonas vaginalis is a protozoan parasite that is responsible for trichomoniasis, the most common non-
viral sexually transmitted infection (STI). Attachment to host cells is likely to play a significant role in the
establishment of infection, yet T. vaginalis receptors on host cells have not been identified. A candidate
molecule for host-pathogen interactions on the surface of T. vaginalis is its lipophosphoglycan (LPG)-like
molecule. In order to study the importance of LPG in infection, mutants with altered LPG were generated by
chemical mutagenesis followed by selection by lectin agglutination. Selected mutants do not bind to the
lectin RCA120 or wheat germ agglutinin, indicating losses of galactose and N-acetyl glucosamine. The
mutant parasites furthermore have clear differences in the monosaccharide composition of their LPG
molecules. Differences in adhesion and cytotoxicity to ectocervical cells suggest that LPG is involved in
parasite-mediated cytotoxicity.     These mutants underscore the importance of T. vaginalis LPG in
establishing infection. Because of the abundance of LPG molecules on the parasite surface, it is possible
that T. vaginalis utilizes host cell lectins as receptors. An S-type lectin found on host cells called galectin-1
has been recently implicated in binding a variety of pathogens. We now show that T. vaginalis binds to
galectin-1 in a lectin-specific manner. T. vaginalis LPG may be the specific molecule that binds to this lectin,
as the LPG mutants do not bind galectin-1. Studies are currently in progress to determine whether T.
vaginalis is capable of utilizing this host cell lectin as a receptor to initiate binding of the parasite to host
cells.
TRICHOMONAS VAGINALIS AND THE FIRST LINE OF HOST DEFENSE

Daniele Dessì, Paola Rappelli, Pier Luigi Fiori
Dept of Biomedical Sciences, Division of Clinical and Experimental Microbiology
University of Sassari, Sassari (Italy)

The establishment of a successful infection by a pathogenic microorganisms usually requires the overcoming
of the front line of host defenses, represented by the innate immune response. The interaction of an invading
microorganism with the cellular component of innate immunity (neutrophils, monocytes/macrophages and
dendritic cells –DCs-) dictates the fate of the infection and the type of adaptive immune response. The
abundance of leukocyte infiltration observed during acute trichomoniasis indicates a key role of innate
immune cells in the first response to T. vaginalis. While the important role of neutrophils in the
proinflammatory response to T. vaginalis infection has already been described, the interaction with
monocytes/macrophages and DCs, which patrol the different districts of the organism acting as sentinels for
invading microbial pathogens, still remains unclear. We investigated the contribution of these cells to the
proinflammatory response to T. vaginalis, which is fundamental in both clearance of infection and
pathogenesis. An in vitro model of macrophage/T. vaginalis interaction showed how the protist elicits the
secretion of inflammatory cytokines (IL-1B, TNF-a, IL-8 but not IL-6) in a dose- and time-dependent fashion.
The presence of Mycoplasma hominis, a bacterium that establishes a biological association with T. vaginalis,
upregulates cytokine secretion. In opposition to mycoplasma-free T. vaginalis, mycoplasma-infected
protozoan cells induce the secretion of IL-12, a cytokine involved in the response to intracellular pathogens.
The same experiments were conducted using paraformaldheyde(PFA)-fixed microorganisms, which induced
the secretion of lower amounts of pronflammatory cytokines as compared to live microorganisms. This
indicates a possible role for trichomonal secreted factors in the inflammatory response by macrophages.
DCs are professional antigen presenting cells that specifically recognize pathogen-derived structures in
peripheral tissues and consequently mature and migrate to lymph nodes to initiate the adaptive immune
response. An in vitro model of T. vaginalis/DC interaction showed that both live and PFA-fixed protozoan
cells induce the maturation of DCs, which subsequently secreted only an anti-inflammatory cytokine, IL-10.
Taken together, these results suggest that the interaction of T. vaginalis with innate immune cells may
strongly influence the fate of protozoan infection. The strong proinflammatory response of macrophages may
contribute to both clearance of infection and damage to vaginal epithelium. The antinflammatory cytokine
milieu produced by DCs after interaction with T. vaginalis is consistent with the clinical presentation of
chronic and asymptomatic T. vaginalis infections and may partly explain the apparent lack of an effective and
protective adaptive immune response.
GENE DUPLICATION AND HORIZONTAL GENE TRANSFER IN TRICHOMONAS VAGINALIS
GENOME EVOLUTION: IDENTIFICATION OF POTENTIAL PATHOGENIC FACTORS
        1       2             2               1              1            1
Noel, C , Lal, K , Thomas, R , Alsmark, UC , Embley, TM and Hirt, R .
1 School of Biology, The University of Newcastle, Newcastle NE1 7RU, UK
2 Department of Zoloogy, The Natural History Museum, Cromwell Rd, London, SW7 5BD, UK.

Investigating the diversity of genes among 4000 EST from Trichomonas vaginalis (G3 strain) we identified
several cases of potential horizontal gene transfers (HGT) and unusual amplification of typical eukaryotic
gene families. These genes represent potentially interesting drug targets to control T. vaginalis and three
gene families were analyzed in more details. Two gene families involving strong candidate HGT, likely
followed by gene duplication, encode potential surface and secreted proteins that represent strong candidate
proteins acting as pathogenic factors. A case of an unexpected large gene family encodes Rab small
GTPase that are known to orchestrate membrane trafficking in model organisms. For each gene family we
present evidence that several genes are transcribed and in some cases are up-regulated during binding to
host proteins of the extracellular matrix further supporting their likely biological relevance.
IRON IN THE VIRULENCE OF TRICHOMONAS VAGINALIS

Rossana Arroyo
Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN
(CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro Zacatenco, C.P 07360, México D.F., México. E-mail:
rarroyo@cinvestav.mx

Iron is an essential nutrient for growth, metabolism, and expression of virulence factors for many pathogens
including Trichomonas vaginalis. In this protozoan, iron concentrations modulate a differential expression of
surface antigens, hydrogenosomal proteins (pyruvate ferredoxin oxidoreductase [PFO], ferredoxin), and
positively regulate the levels of cytoadherence and relocalization of the adhesins AP65, AP51, AP33, AP23,
as well as the new adhesin AP120 recently identified and characterized. T. vaginalis has multiple proteolytic
activities many of the cysteine type (CPs), some of which are considered virulence factors such as CP30 and
CP62 involved in cytoadherence, and CP65 and CP39 in cytotoxicity. Interestingly, the proteolytic activities
from the 30-kDa region of trichomonad extracts belong to papain and legumain families from two different
clans, CA and CD, respectively. These proteinases are differentially regulated by iron at the enzyme activity
level as demonstrated by 2-D zymograms. Iron also up-regulates the proteolytic activity of unidentified CPs
over the C3b complement component deposited on the parasite surface. Although, the mechanism of iron
regulation upon CP activity still unknown, it has been found that iron modulates the transcription of some of
the CP genes; i.e., tvcp4 gene is better transcribed in presence of iron, whereas, tvcp12 is in the absence of
it. Likewise, iron also negatively affects other genes such as the fibronectin-like flp-1 and flp-2 and the
tvcp65, the gene that encodes for CP65. All these data show that iron concentrations could have a dual
effect in the virulence of T. vaginalis, suggesting a very fine-tuning mechanism of gene expression regulation
mediated by iron, probably through some iron regulatory elements like the one described by Tai group, which
is found at the 5’ untranslated regions of the positively regulated ap65 gene.
GIARDIA - HOST CELL INTERACTIONS: IT TAKES TWO TO TANGO
                1                              2             2                    1,2, *
Emma Ringqvist , Katarina Roxström-Lindquist , Daniel Palm and Staffan Svärd
1 Department of Cell and Molecular Biology, Uppsala University, SE-75124 Uppsala, Sweden
2 Department of Parasitology, Microbiology and Tumor Biology Center, Karolinska Institutet, SE-17177
Stockholm, Sweden.

The parasitic protozoan Giardia lamblia is a worldwide cause of diarrhea, but the mechanism of disease
remains elusive. The parasite colonizes the small intestinal epithelium, known to be a sensor for the
presence of enteric pathogens, without invading or causing severe inflammation.
Excretory-secretory products of pathogenic microbes often play important roles in host-parasite interactions.
Using proteomics approach, we have identified the major proteins in the culture supernatant after in vitro
interaction between Giardia lamblia and the human intestinal epithelial cell lines Caco-2 and Ht-29. The
importance of the secreted proteins during host-parasite interactions has been further studied.
We have also investigated the epithelial cell response to G. lamblia by oligonucleotide microarrays. A large
number of genes displayed changed expression patterns, showing the complexity of the interaction between
G. lamblia and intestinal cells. A novel chemokine profile (CCL2, CCL20, CXCL1, CXCL2 and CXCL3) was
induced, different than the response induced by enteric pathogens causing intestinal inflammation. Several
genes involved in stress regulation changed their expression. These findings indicate that the intestinal
epithelium senses the G. lamblia infection and this is important for induction of innate- and adaptive
immunity. The induced stress response can be important in the pathogenesis.
        Monday 19th September 2005

                14:00 – 16:00

            BIOCHEMISTRY
Chairpersons: Louis Tielens and Ching C. Wang
ENERGY GENERATION IN ANAEROBIC PROTISTS

Aloysius G.M. Tielens and Jaap J. van Hellemond
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, PO Box
80176, 3508 TD Utrecht, The Netherlands

Numerous protists exist that can function without oxygen. Organisms that are able to function without oxygen
as terminal electron acceptor have to maintain redox balance without aerobic respiration. Hence, the
reduced cofactors produced by the catabolic pathways have to be oxidized by an alternative process. This
presentation will be devoted to adaptations to hypoxia involving fermentation pathways where an
endogenous electron acceptor is used. Protists using an endogenous electron acceptor other than oxygen
can be divided into two classes: those that use for the production of ATP simple fermentations in the cytosol
only, and those that use more complex anaerobic processes in specialized ATP-producing organelles, i.e.
anaerobic mitochondria or hydrogenosomes.
        Permanently anoxic functioning eukaryotes are always unicellular and live in hypoxic environments
such as marine or freshwater sediments, or the rumen and intestinal tracts of their host. These protists
produce ATP via anaerobic fermentations, and they are divided into two groups, designated type I and type
II, depending on the absence or presence of hydrogenosomes, respectively. Type I amitochondriate protists
do not possess ATP-producing organelles and are fully dependent on substrate-level phosphorylations for
ATP production, which occur exclusively in the cytosol. Giardia and Entamoeba, human intestinal parasites,
are the best studied representatives of these Type I anaerobic protists. Type II anaerobic organisms, on the
other hand, do contain specialized organelles, which produce ATP and hydrogen, and are therefore called
hydrogenosomes. Hydrogenosomes are double membrane bound organelles, clearly related to mitochondria
but they lack oxidative phosphorylation and produce ATP by substrate level phosphorylation only.
        Next to these various types of anaerobic organisms, some protists exist that can use oxygen when
present, but can also live in its absence, as they contain specially adapted mitochondria. Euglena is a well
studied example containing such mitochondria. These facultative anaerobes possess a mitochondrion that in
the presence of oxygen produces ATP via oxidative phosphorylation with oxygen as final electron acceptor,
but can also use oxidative phosphorylation in the absence of oxygen via a specially adapted electron
transport chain .
        Metabolic differences between the various types of hydrogenosomes and mitochondria, and
between the various cytosolic adaptations to hypoxia in protists will be discussed.
DIVERSITY AND EVOLUTION OF ANAEROBIC ENERGY-GENERATING ORGANELLES
                         1*                 2
Johannes H.P. Hackstein and Nigel Yarlett
1
Dept. Evolutionary Microbiology, Fac. Science, Radboud University Nijmegen, Nijmegen, The Netherlands
    2
and Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, New York,
USA

Hydrogenosomes are organelles, approximately 1-2 micrometer in size, that compartmentalize the terminal
reactions of the anaerobic cellular energy metabolism. They were first described in the parabasilid flagellate,
Tritrichomonas foetus, in a seminal publication by Lindmark and Müller (1973) as subcellular compartments
that produce hydrogen and ATP. Since this time hydrogenosomes or variations of them have been described
in quite a number of rather different unicellular eukaryotes adapted to microaerobic or anoxic environments
(Roger 1999; Yarlett 2004). Several researchers considered these organelles as variations of mitochondria
adapted to anaerobic environments (Embley et al. 2003), but it is still a matter of debate whether or not
these ancestral “mitochondria” had an aerobic metabolism using oxygen as a terminal electron acceptor - or
whether these organelles functioned anaerobically, and, consequently, produced hydrogen like present-day
hydrogenosomes (Tjaden et al. 2004). Moreover, since most of these organelles lack a genome studying
their evolutionary relationships frequently has to rely on circumstantial evidence. Comparisons between the
various types of hydrogenosomes reveal substantial differences in structure and function and reinforce the
conclusion that hydrogenosomes are the products of evolutionary tinkering, modifying – most likely – one
and the same ancestral organelle. This interpretation is supported by the discovery of a true missing link: an
anaerobic mitochondrion that makes hydrogen (Boxma et al. 2005).



Boxma B et al. (2005) Nature 434: 74-79
Embley TM et al. (2003) IUBMB Life 55: 387-395.
Lindmark DG & Muller M (1973) J Biol Chem 248: 7724-7728
Roger AJ (1999) Am Naturalist 154: S146-S163
Tjaden J et al. (2004) Mol Microbiol 51: 1439-1446
Yarlett N (2004) Microbiology-SGM, 150:127-129




* with contributions by Brigitte Boxma, Rob M. de Graaf, Georg W.M. van der Staay, Theo A. van Alen,
Guenola Ricard, Toni Gabaldón, Angela H.A.M. van Hoek, Seung Yeo Moon-van der Staay, Werner J.H.
Koopman, Jaap J. van Hellemond, Aloysius G.M. Tielens, Thorsten Friedrich, Marten Veenhuis, Martijn A.
                                                                   th
Huynen and the consortium supported by the European Union 5 framework grant “CIMES” (QLK3-2002-
02151).
CATALYTIC       MODULE        OF    MITOCHONDRIAL-TYPE             RESPIRATORY         COMPLEX        1   IN
TRICHOMONAS VAGINALIS HYDROGENOSOMES
         1            2                  1               1                2               1
Ivan Hrdy , Robert Hirt , Lucie Bardonova , Pavel Dolezal , Martin Embley , Jan Tachezy
1
Charles University, Prague, Czech Republic
2
School of Biology, The University of Newcastle, Newcastle upon Tyne, United Kingdom

Trichomonad hydrogenosomes use pyruvate and malate as carbon source in the oxidative reactions
connected to substrate-level synthesis of ATP. Pyruvate:ferredoxin oxidoreductase (PFOR), the typical
enzyme of hydrogenosomes, uses ferredoxin as electron acceptor in oxidative decarboxylation of pyruvate
leading to formation of acetyl coenzyme A. Ferredoxin is reoxidized, under anaerobic conditions, by
hydrogenase which forms molecular hydrogen.             First step in malate catabolism is its oxidative
decarboxylation to pyruvate and carbon dioxide by malic enzyme, the most abundant hydrogenosomal
protein that uses NAD as electron acceptor. Since hydrogenosomal membrane is impermeable to pyridine
nucleotides, the question arises as to how reduced NADH is reoxidized. The NADH:ferredoxin
oxidoreductase activity, that could recycle NAD, was identified in hydrogenosomes long time ago, but the
nature of the enzyme remained elusive. We have isolated this protein and identified its coding sequences in
T. vaginalis EST and genome databases.        The enzyme is heterodimeric protein that is able to reduce a
number of artificial as well as naturally occurring electron acceptors, including coenzyme Q1 and trichomonad
ferredoxin, the latter apparently with highest affinity. The protein also participates in PFOR-independent
activation of antitrichomonad drug metronidazole. Sequence analysis revealed that the two subunits are
homologous to NuoF/51 kDa and NuoE/24 kDa subunits of catalytic, electron-input module of multi-subunit
respiratory complex I from aerobic bacteria and mitochondria. Detailed phylogenetic analysis indicated that
T. vaginalis NuoF homologue is more closely related to mitochondrial orthologues from other eukaryotes
rather than to bacterial complex I subunit. Recruitment of mitochondrial-type protein into ferredoxin-
dependent hydrogen-producing pathway lends further support to common ancestry of mitochondria and
hydrogenosomes.
AMINO ACID METABOLISM IN TRICHOMONAS VAGINALIS
                     a                    a                      b
Graham H. Coombs , Gareth D. Westrop , Jeremy C. Mottram
a
Division of Infection & Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Joseph
Black Building, Glasgow G12 8QQ, UK
b
Wellcome Centre for Molecular Parasitology, University of Glasgow, The Anderson College, Glasgow G11
6NU, Scotland, UK

Analyses of the genome database for Trichomonas vaginalis G3 have allowed predictions of amino acid
metabolic pathways that operate in the parasite. Several unusual features have become apparent, some
already revealed from experimental analyses. Important differences from the metabolic pathways operating
in humans include the metabolism of cysteine. Production of recombinant enzymes has allowed analysis of
the specificities of the key enzymes, which will be reported. Of particular note is the finding that the synthesis
of cysteine depends upon an unusual cysteine synthase. The level of cysteine synthase in the parasite is
regulated according to need, such that parasites growing in an environment rich in cysteine have low activity,
whereas exposure to propargylglycine results in elevated cysteine synthase activity. As humans lack
cysteine synthase, this parasite enzyme could be an exploitable drug target.
CERAMIDE ENDOCYTOSIS AND METABOLISM BY AN AMITOCHONDRIATE PROTOZOAN,
GIARDIA LAMBLIA

Siddhartha Das, Yunuen Hernandez, Jaime Chapoy, Stephen B. Aley
Department of Biological Sciences, University of Texas at El Paso, El Paso, TX.

The amitochondriate protozoan, Giardia lamblia, is a major cause of waterborne enteric disease worldwide.
One of the fascinating aspects of giardial biology is its limited ability to synthesize membrane lipids de novo.
We have shown earlier that Giardia generates parasite-specific phospho- and glycolipids by remodeling
reactions. The goal of the present investigation is to understand the phenomenon of ceramide uptake and
trafficking by Giardia, since ceramide is an important component of all classes of sphingolipids. Results
suggest that this protozoan internalizes ceramide by actin-mediated endocytic processes and spontaneously
targets this compound into ER/perinuclear membranes with the help of microtubule filaments. Furthermore,
the extensive co-localization of Bodipy-ceramide with clathrin but not dynamin-positive vesicles or lipid rafts
indicates that ceramide is transported mostly by clathrin-mediated endocytosis. Psi-BLAST searches of the
Giardia genome failed to identify candidates for most genes in the sphingolipid metabolic pathway,
consistent with the apparent lack of ceramide synthetic ability by Giardia, although a potential homolog for
serine-palmitoyltransferase-2 was noted. We found that D, L- -fluoropalmitic acid, which blocks sphingolipid
synthesis, reduced the growth of the parasite in culture. These studies suggest the presence of well-
orchestrated transport machineries that coordinate the efficient transport and targeting of exogenous
ceramide in this protozoan. Our results also suggest that ceramide/sphingolipid is important for the growth
and survival of Giardia, and should be considered as a target for developing new drugs against this
waterborne pathogen.
THE TWO INHIBITORS OF CYSTEINE PROTEASES, AMOEBIASIN-1 AND -2,

IN TROPHOZOITES OF ENTAMOEBA HISTOLYTICA

Mirela Šarić, Sabine Riekenberg, Anke Harsman, Henning Scholze*
University of Osnabrueck, Dept. Biology/Chemistry, 49069 Osnabrueck, Germany

According to data of the recently published Entamoeba Genome Project, Entamoeba histolytica is equipped
with more than 50 cysteine protease genes, part of which are presumed to be involved in different aspects of
pathogenesis. So far, nothing is known concerning regulation of these enzymes. Generally, protease activity
is controlled on several levels of cellular organization reaching from protease gene expression up to the
synthesis of protease-inhibiting proteins. In addition to a gene for a serpin-like protease inhibitor, the genome
of E. histolytica contains two genes (abbreviated as Ehicp1 and Ehicp2) encoding cysteine protease
inhibitors, both having homology to chagasin, the prototype of a new protease inhibitor class first discovered
in the parasitic protozoon Trypanosoma cruzi. Sequence alignment of the amebic inhibitors, amoebiasin-1
and amoebiasin-2, and of homologous inhibitors together with hypothetical proteins from various pathogenic
prokaryotes and eukaryotes and construction of a phylogenetic tree suggests that these proteins are not a
result of simple gene duplications, but are rather products of several horizontal gene transfers. We have
proved the expression of both inhibitor-encoding genes in the trophozoites by Western and Northern blotting.
In an indirect immunofluorescence, amoebiasin-1 showed a fine dotted distribution across the cytoplasm,
which does not coincide with the location of the major amoebic cysteine proteases EhCP1 and EhCP5. Both
recombinant amoebiasins inhibited cysteine protease activity in a dose-dependent manner, with equilibrium
dissociation constants (Ki) in the picomolar to µmolar range. As predicted for chagasins, secondary structure
calculations of amoebiasin revealed mainly β-sheet structure only interrupted by loops comprising the three
conserved motifs. Particular importance concerning the nature of protease inhibition should be attributed to
the proline residues in two of the conserved motifs. In an inhibitory test using a synthetic peptide with the
sequence of the first conserved motif of amoebiasin-1, GNPTTGF, we indeed observed inhibition of cysteine
protease activity. This inhibitory effect is abolished, when Pro is exchanged for Ala, a fact that emphasizes
the particular role of proline in contacting the active site of the target protease. A similar model could apply to
the third conserved motif, YXRPW. This sequence strongly resembles the second hairpin loop in cystatins,
which suggests a convergent evolution of cysteine protease inhibition as a general principle comparable to
the common mode of peptide bond cleavage mechanisms by serine and cysteine proteases.
CRYPTOSPORIDIA INFECTION ALTERS HOST CELL POLYAMINE LEVELS

Nigel Yarlett* and Mary Morada
Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, New York, NY
10038, USA

Polyamine metabolism by Cryptosporidium parvum occurs via two routes, one utilizing a “forward pathway”
via a plant-like pathway involving arginine decarboxylase; and the other a “reverse pathway” involving
spermidine:spermine N-acetyltransferase (SSAT) and polyamine oxidase (PAO). The parasite actively
transports polyamines, particularly spermine, from the extracellular milleau. Our studies lead us to believe
that in situ the uptake and back conversion of polyamines provides the major source of these critical
nutrients to the parasite. The availability of intestinal polyamines to the parasite has not previously been
explored. In this study we demonstrated that C. parvum can upregulate polyamine metabolism by the host
cell resulting in increased expression of SSAT, with minimal effect upon the expression of PAO. SSAT is the
rate-limiting step in polyamine backconversion by eukaryotic cells, hence the result of increased activity of
SSAT would cause increased production of acetylspermine and acetylspermidine for transport out of the host
cell. This overproduction and export of polyamines by the host cell results in an abundant source of these
amines that can then be used by the parasite for growth and cell division. Agents that blocking these events,
such as conformationally restricted polyamine analogues may be an effective and novel mechanism to cure
this disease.


Funded by the National Institutes of Health AI49785 (NY).
       Tuesday 20th September 2005

               9:00 – 10:40

               Evolution
Chairpersons: Andreas Brune and David Lloyd
THE     CARDIOLIPIN-CONTAINING               MITOCHONDRIA-LIKE             ORGANELLES           OF     GIARDIA
INTESTINALIS (SYN.LAMBLIA, DUODENALIS)

David Lloyd, Anthony J. Hayes, Michael P. Turner, Deborah Cole, Michael O’Reilly and Jayne Ellis*
Microbiology (BIOSI1), Cardiff University P.O. Box 915 Cardiff, CF10 3TL, Wales UK. * Unipath, Priory Park,
Bedford MK44 3UP,UK.

Organelles that show redox activities and other characteristics of mitochondria have been demonstrated in 5
strains of Giardia intestinalis (syn. lambia,duodenalis). Incubation of the trophozoite life-cycle stage in growth
medium with a tetrazolium salt (e.g. triphenyl-tetrazolium chloride) results in the deposition of insoluble
formazan reduction product within numerous organelles distributed peripherally in the organism in the sub-
pellicular zone. A similar location has been observed for binding of 10-N nonylacridine orange bromide, a
fluorophore specific for cadiolipin of inner mitochondrial membranes. Both these in vivo mitochondrial
labeling procedures resulted in organisms with short-term unimpaired motility. Fluorescence of sub-pellicular
vacuoles after reaction with fluorescent antibodies specific for clathrin-containing organelles or for a
peipheral vacuole protein, show distinctive distribution in confocal images: these are quite different from
                                                                                            6
those of the mitochondria-like organelles. A particulate fraction sedimenting at 6.10 g min from gently-
disrupted organisms and containing 72% of the pyruvate:methyl viologen oxido-reductase, 68% of the
NADPH-FMN oxido-reductase and 57% of the NADH-FMN oxido-reductase, found in the homogenate,
showed oxygen consumption. Pyruvate and the two reduced nicotinamide nucleotides were oxidized by this
fraction and substrate oxidations were stimulated by FAD. Tetrazolium reduction also occurred in a gradient-
purified fraction containing these particles and some of them showed inner and outer membranes in high
magnification transmission electron micrographs. The organism’s parasitic life-style in a low O2 environment
has led to loss of some characteristic mitochondrial structural features and energy-conserving mitochondrial
functions.
MOLECULAR             PHYLOGENY VERSUS            SYSTEMATICS          BASED      ON     MORPHOLOGICAL
CHARACTERS IN PARABASALIDS: CONFLICT OR CONGRUENCE?
                      1                       1                  2                        2
Christophe NOËL , Fabienne DUFERNEZ , Satoko NODA , Shigeharu MORIYA , Pilar DELGADO-
               3                    1                 2                   2                       1
VISCOGLIOSI , Monique CAPRON , Toshiaki KUDO , Moriya OHKUMA , and Eric VISCOGLIOSI
1                                                 2
Inserm U547, Institut Pasteur of Lille, France; Environmental Molecular Biology Laboratory, RIKEN, and
                                          3
JST-PRESTO, Wako, Saitama, Japan; Department of Water and Environment, Institut Pasteur of Lille,
France.

The current taxonomy of Parabasalia (or parabasalids) is based on morphological characters, mostly linked
to the structure and development of the cytoskeleton. More than 80 genera and 400 species were identified
so far and separated into two classes: the Trichomonadea (or trichomonads) and the Hypermastigea (or
hypermastigids). The Trichomonadea have been subdivided into four families: Monocercomonadidae,
Trichomonadidae, Devescovinidae, and Calonymphidae whereas the hypermastigids were divided into three
orders. The Monocercomonadidae, which includes species exhibiting a rudimentary cytoskeleton, have been
thought to occupy a basal position in the parabasalid tree. The other trichomonad families, with their more
complex cytoskeleton, were thought to be derived from monocercomonad ancestors. Finally, the
hypermastigids, which are characterized by hyperdevelopment of the cytoskeleton and multiplication of
flagella, were positioned at the apex of the parabasalid tree, evolving from the simpler types.
In recent years, phylogenetic studies of parabasalids have begun to use molecular sequence data, primarily
SSU rRNA gene sequences. In Trichomonadidae and Monocercomonadidae, many members have been
cultivated and thus well-studied phylogenetically. In contrast, hypermastigids as well as devescovinids and
calonymphids are all found exclusively in the hindgut of lower termites and wood-eating cockroaches and are
resistant to cultivation, presenting a serious obstacle for taxonomic studies of these microorganisms using
molecular means. Recently, however, SSU rRNA gene sequences from parabasalid symbionts were
obtained by PCR amplification of whole gut fauna. This has led to the accumulation of a large number of as
yet unidentified sequences. In some studies, the corresponding organisms from which the SSU rRNA gene
sequences were derived have been identified by whole-cell in situ hybridizations with sequence-specific
probes. Alternatively, sequence data were also obtained by amplifying gene sequences from a small
population of some uncultivated symbionts physically isolated under microscopy.
From a broad phylogeny including all the identified parabasalid sequences, several salient points will be
discussed such as the polyphyly of both classes of parabasalids and all trichomonad families. Moreover, the
root of parabasalids has been examined but could not be discerned accurately. Despite congruencies only
observed at some lower and intermediate taxonomic levels, the molecular data conflict with the established
systematics based on a limited number of morphological characters. These data indicate a necessary
revision of the parabasalid systematics that should take into account both molecular and morphological data
in the near future.
THE FIBROLYTIC GENES IN RUMEN CILIATES WERE ACQUIRED BY LATERAL GENE
TRANSFER

NEIL R. McEWAN
Rowett Research Institute, Aberdeen, AB21 9SB, Scotland

Ruminants represent one of the most species-rich and flourishing mammalian taxa. Their evolutionary
success appears to be due to a complex foregut differentiation – the rumen. This organ hosts an extremely
complex microbial community of methanogenic archaea, eubacteria, chytridiomycete fungi and ciliates,
which enables the host to break down plant-polymers such as cellulose and hemicelluloses, and to use the
resulting monomers, the microbial fermentation products, and the microorganisms themselves as a food
source.
Ciliates are the most numerous and conspicuous eukaryotes in the rumen, accounting for up to 50% of the
microbial biomass present, but only a few large ciliate species are able to engulf plant fibres. Here it is
shown, that these larger ciliate species possess a broad spectrum of fibrolytic enzymes, which have been
acquired by lateral gene transfer from rumen bacteria. The genes of the rumen ciliates have a highly biased
codon usage pattern and make use of only a sub-population of the codons which are available. Analysis of
the codon usage of the genes encoding fibrolytic enzymes strongly suggests that these genes were acquired
“recently” since the codon preferences exhibit an intermediary state of ameliorization.


The contributions of Johannes Hackstein, Guenola Ricard and Martijn. Huynen (Radboud University
Nijmegen), Derek Gatherer (MRC Virology Unit, Glasgow), Freda McIntosh, Nadine Thomas, Nancy Nelson,
Jeremy Pontvianne and Anne-Elise Chmielowiec (Rowett Research Institute), Jean-Pierre Jouany, Eli
Nsabimana, Didier Macheboeuf and Diego Morgavi (I.N.R.A.), Tadeusz Michalowski and Krzysztof
Wereszka (Kielanowski Institute of Animal Physiology and Nutrition) and Jamie Newbold (University of
Wales)
This work was in part funded as part of the EU Framework V Quality of Life and Management of Living
Resources Research Programme grants CIMES (QLK3-2002-02151) and ERCULE (QLRI-CT 2000 01455).
The Rowett Research Institute receives funding from SEERAD.
GIARDIA MITOSOMES: VESTIGIAL ANAEROBIC MITOCHONDRIA?

Danai Zourmpanou, Gloria Leon-Avila, Pelagia Deriziotis, Mark van der Giezen & Jorge Tovar
School of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, United Kingdom

Highly derived mitochondrion-related organelles of somewhat heterogeneous morphology and predicted
function exist in a range of anaerobic protists including Giardia intestinalis, Entamoeba histolytica,
Trachipleistophora hominis and Cryptosporidium parvum. Originally described in Entamoeba histolytica as
mitosomes or cryptons, these organelles are promising model systems to establish the minimal requirements
for the maintenance of a self-replicating endosymbiosis-derived organelle. Giardia mitosomes participate in
the biosynthesis of iron-sulphur (FeS) clusters, a physiological role they share with mitochondria and
hydrogenosomes (another type of mitochondrion-related organelle). Most luminal mitochondrial and
hydrogenosomal proteins are imported into their respective organelles via positively charged and cleavable
amino-terminal targeting presequences. Of the known giardial mitosomal proteins only IscU and ferredoxin
(Fd) contain putative organelle targeting presequences. Using GFP fusion proteins expressed in transgenic
parasites along with deletion and substitution experiments, we have investigated the functionality of such
putative mitosomal targeting signals in vivo. We found that both amino-terminal presequences are essential
and sufficient for mitosome targeting and show they are cleaved off upon organelle import. Efficient targeting
into mouse mitochondria of a passenger protein fused to a mitosomal targeting presequence demonstrated
that conserved protein import mechanisms operate in mammalian mitochondria and Giardia mitosomes. It
has been reported that Giardia contains specialized membranes with electron transport and membrane
potential generating functions. Co-localisation experiments using mitosomal marker proteins and rhodamine
123 staining or formazan precipitation clearly indicated that mitosomes lack a detectable membrane potential
and are not involved in CTC-salt reduction. Thus, the precise nature of formazan precipitation and of the
rhodamine-permeable membranes of Giardia remains to be determined. Overall, our data support the view
that Giardia mitosomes represent vestigial anaerobic mitochondria.
HORIZONTAL GENE TRANSFER IN ENTAMOEBA HISTOLYTICA: EVIDENCE FOR AN
IMPORTANT ROLE OF RECENTLY ACQUIRED GENES FROM PROKARYOTIC ORIGINS
            1                    2          3         1                 1
Alsmark, UC , Sicheritz-Ponten, T , Foster, P , Hirt, RP and Embley, TM
1 School of Biology, The University of Newcastle, Newcastle NE1 7RU, UK
2 Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, DK-2800
Lyngby
3 Department of Zoloogy, The Natural History Museum, Cromwell Rd, London, SW7 5BD, UK.

Horizontal gene transfer (HGT) has been postulated to play an important role in prokaryotic genome
evolution. More recently data also suggested that HGT, outside the well-established HGT derived from
endosymbiotic organelles (mitochondria and plastids), could play an important role in microbial eukaryotes,
possibly facilitated in phagocytic taxa that feed on prokaryotes. We have analyzed the complete proteome of
Entamoeba histolytica (9938 proteins) to identify candidate HGT genes by producing alignments and
phylogenetic analyses for all proteins with the bioinformatic tool PyPhy. A primary screen based on Blast
reports and p-distance trees was used to identify potential HGT cases and a secondary, more sophisticated,
phylogenetic screening was used to establish the strongest case of HGT. A total of 96 strong cases of HGT
were established and these have clearly affected important aspect of the biology of Entamoeba with in
particular expansion of its metabolic capabilities. Both the methodology used and biological inferences will
be presented.
Tuesday 20th September 2005

        11:00 – 12:00

Drugs and drugs targets
 Chairperson: Jaroslav Kulda
ENCYSTMENT AS A TARGET FOR DRUG DESIGN

Edward L. Jarroll
Department of Biology, Northeastern University, 360 Huntington Ave. Boston, MA, 02115 USA

Flagellates, amoebae, ciliates, coccidia, and several species of microsporidia infect the human intestinal
tract. Many of these protozoa exhibit simple asexual life cycles while others exhibit complex life cycles with a
sexual and asexual stages. Yet, all of these protozoa have life cycle strategies that involve leaving one host
after encysting to infect another. Encysted forms usually exit the host in feces and enter the next host orally.
What exactly triggers encystment and excystation in these protozoa is not completely understood, but
studies of these processes are underway. These processes are certainly better understood in Giardia than
in any of the other protozoa infecting human intestines. During Giardia encystment, metabolic events related
to the formation of leucine rich cyst wall proteins (ca. 37%) and a β (1, 3) N-acetylgalactosamine
homopolymer (ca. 63%) that form cyst wall filaments occurs. Early in encystment, genes encoding cyst wall
proteins (CWP) and UDP-GalNAc synthesizing enzymes are expressed.                CWPs are detectable in the
resulting cyst wall filaments along with the GalNAc polysaccharide. Five of the six GalNAc synthesizing
enzymes are cytosolic, while one is particle-associated, and all six are inducible. Genes for the cytosolic
enzymes in this inducible pathway have been cloned and sequenced and all of them are apparently induced
during encystment. Activity of the particle-associated “cyst wall synthase” has been detected and partially
purified and characterized, but this enzyme has not yet been cloned.
        Given that resistance to antiprotozoal drugs exists and is likely to increase and given that currently
no reliable treatments exist for some of these infections, efforts to find new targets for chemotherapy must
be continued. In the case of cyst forming pathogenic protozoa, one such target might be encystment
pathways and cyst wall assembly. Information is increasing on protozoan encystment, and we can begin to
answer the question of whether targeting it for chemotherapy is a viable drug strategy. We know that the
encystment process in Giardia offers 1) unique proteins, 2) unique polysaccharides and 3) unique enzymes
making all potentially interesting targets for chemotherapeutic attacks. For example, the development of β
(1, 3) glucan synthase inhibitors is receiving attention in fungal systems because these inhibitors potentially
target fungal cell wall structure. Among these drugs are the cyclic lipopeptides echinocandins and the
liposaccharides papulacandins.
        Portions of this work were supported by NIH Award AI4775.
KALETRA - AN EFFECTIVE ANTI-TRICHOMONAL AND ANTI-GIARDIAL AGENT

Jacqui Upcroft, Linda Dunn and Peter Upcroft
Queensland Institute of Medical Research, 300 Herston Rd, Brisbane, Queensland 4006, Australia

HIV protease inhibitors were first introduced in the mid 90s. Currently, there are five approved by the FDA for
the treatment of HIV infection. These include Invirase (saquinavir), Norvir (ritonavir) and Crixivan (indinavir)
and more recently Abbott introduced Kaletra, a combination of lopinavir (poorly soluble), Norvir (added to
metabolically protect lopinavir) and solubilising agents. Since many individuals currently take these
medications and these same individuals are especially at risk for sexually transmitted infections (other than
HIV) including Trichomonas vaginalis, Entamoeba histolytica (recently emerging in gay communities in
Australia and Japan [1]), and Giardia duodenalis (transmissible amongst homosexual men [2]), it is of
importance to know the effect of these drugs on the range of organisms with which they come in contact. In
addition, kaletra was recently shown to be effective against Plasmodium [3].

        We tested saquinavir, ritonavir, lopinavir and kaletra against G. duodenalis, T. vaginalis and E.
histolytica in vitro in our standard anaerobic drug susceptibility assay [4]. The combination drug kaletra was
remarkably effective against Giardia with the individual components (ritonavir and lopinavir) exerting various
effects. Trichomonas was less susceptible to kaletra than Giardia with marked strain variation but the effect
of kaletra on Entamoeba was unconvincing.

        Further investigation into the effects of these drugs on Giardia, Trichomonas and Entamoeba is
warranted considering the prevalence of these organisms in association with HIV incidence, the occurrence
of metronidazole resistant Giardia and Trichomonas and the fact that these drugs are already widely
approved for administration to both adults and children.


Acknowledgments: Lopinavir (cat # 9481) was obtained through the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID (contributor), NIH.


    1. Ohnishi K, Kato Y, Imamura A, Fukayama M, Tsunoda T, Sakaue Y, Sakamoto M, Sagara H.
        Present characteristics of symptomatic Entamoeba histolytica infection in the big cities of Japan.
        Epidemiol Infect 2004;132:57-60.
    2. Pakianathan     MR,    McMillan    A.   Intestinal   protozoa   in   homosexual   men    in   Edinburgh.
        Int J STD AIDS. 1999;10:780-4.
    3. Skinner-Adams TS, McCarthy JS, Gardiner DL, Hilton PM, Andrews KT. Antiretrovirals as
        antimalarial agents. J Infect Dis. 2004;190:1998-2000.
    4. Upcroft JA, Upcroft P. Drug susceptibility testing of anaerobic protozoa. Antimicrob Agents
        Chemother. 2001;45:1810-4.
IDENTIFICATION OF A SUBVERSIVE SUBSTRATE OF TRICHOMONAS VAGINALIS PURINE
NUCLEOSIDE PHOSPHORYLASE AND THE CRYSTAL STRUCTURE OF THE ENZYME-
SUBSTRATE COMPLEX
           1                2                 2               1                 2
Yang Zang , Wen-Hu Wang , Shaw-Wen Wu , Steven Ealick and C. C. Wang
1                                                                                               2
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853 and Department of
Pharmaceutical Chemistry, University of California, San Francisco, CA 94143

        The survival of Trichomonas vaginalis depends on effective purine salvage from its living
environment. One of the pivotal enzymes in the pathway, purine nucleoside phosphorylase (TvPNP), shows
physical properties and substrate specificities similar to those of the high molecular mass bacterial PNPs but
differing from those of human PNP. A nontoxic nucleoside analogue, 2-fluoro-2’-deoxyadenosine (F-dAdo)
was recently identified as a “subversive substrate” for TvPNP. Phosphorolysis by TvPNP of F-dAdo, which is
not a substrate for human PNP, releases highly cytotoxic 2-fluoroadenine (F-Ade). In vitro studies showed
that both F-dAdo and F-Ade exert similarly strong inhibition of T. vaginalis growth with estimated IC50 values
of 106 and 84 nM, respectively, suggesting that F-dAdo might be useful as a potential chemotherapeutic
agent against trichomoniasis.     To understand the basis of TvPNP specificity, the structure of TvPNP
complexed with F-dAdo, 2-fluoroadenosine, formycin A, adenosine, inosine or 2’-deoxyinosine were
determined by X-ray crystallography with resolutions ranging from 2.4 to 2.9 Å. These studies showed that
the quaternary structure, monomer fold and active site are similar to those of Escherichia coli PNP. The
principal active site difference is at Thr-156, which is alanine in E. coli PNP. In the complex of TvPNP with F-
dAdo, Thr-156 causes the purine base to tilt and shift by 0.5 Å as compared with the binding scheme of F-
dAdo in E. coli PNP. Therefore, the structures of the TvPNP complexes suggest opportunities for further
improved subversive substrates beyond F-dAdo.
        Tuesday 20th September 2005

                12:00 – 13:00
        Selected presentations
Chairpersons: Robert Hirt and Eric Viscogliosi
CHARACTERIZATION OF CYTOSOLIC FE-S CLUSTER ASSEMBLY UNDER ANAEROBIC
CONDITIONS IN ENTAMOEBA HISTOLYTICA

Vahab Ali, Kumiko Nakada-Tsukui, and Tomoyoshi Nozaki
Dept. of Parasitology, Gunma University Graduate School of Medicine, Maebashi, Japan

Iron-sulfur (Fe-S) clusters are cofactors of proteins probably present in all living organisms and play various
important roles for survival of the cell. Three independent systems are known for Fe-S cluster assembly, NIF
(nitrogen fixation), ISC (iron-sulfur-cluster), and SUF (mobilization of sulfur). Entamoeba histolytica
possesses various Fe-S proteins, including ferredoxin and pyruvate:ferredoxin oxidoreductase, which play
indispensable roles in energy metabolism and electron transport. E. histolytica has a simplified and non-
redundant NIF-like system for the Fe-S cluster formation, composed of a catalytic component, NifS and a
scaffold component, NifU. This is the first case of the presence of NIF-like system in eukaryotes. NIF-like
system is not present in other anaerobic parasitic protozoa including Giardia and Trichomonas where iron-
sulfur cluster (ISC) system localized in mitosomes or hydrogenosome, respectively plays a homologous role.
An ectopic expression of the EhNifS and EhNifU genes successfully complemented growth defect of an
Escherichia coli strain, in which both the isc and suf operons were deleted, under anaerobic, but not aerobic
condition, suggesting that EhNifS and EhNifU are necessary and sufficient for the Fe-S cluster formation of
non-nitrogenase Fe-S proteins under anaerobic conditions. In the present work, we attempted to determine
factors that are absent in E. histolytica, and thus responsible for the oxygen sensitivity. We introduced
individual genes present in the operon of the bacterial ISC system including an Hsp-70 type- molecular
chaperone (hscA) and a co-chaperone (hscB) of E. coli, together with amebic NifS and NIfU. While
coexpression of these chaperones did not rescue growth defect under aerobic conditions with NifS and NifU,
these molecular chaperones conferred oxygen resistance. We also produced the amebic transformants
overexpressing epitope-tagged or GFP-fused NifS and NifU. These proteins are predominantly localized in
the cytoplasm as confirmed by cellular fractionation and immunoflourescence. The NifS-overexpressing
amoebas contained a 2-3-fold higher cysteine desulfurase activity. Expression of cysteine synthase and
methionine γ-lyase which also possess cysteine desulfurase activity was also slightly down-regulated. We
are currently examining changes in the expression and activities of various Fe-S proteins and sensitivity of
H2O2 of these transformants.
PUTATIVE        [Fe]-HYDROGENASE           MATURASES          IN    THE     HYDROGENOSOMES               OF
TRICHOMONAS VAGINALIS
            a               b                        a              b                  a
Simone Pütz , Pavel Dolezal , Gabriel Gelius-Dietrich , Jan Tachezy and Katrin Henze
a
Institut für Botanik III, Heinrich Heine Universtität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf,
Germany
b
Department of Parasitology, Faculty of Science, Charles University, Vinicna 7, Prague 2 128 44, Czech
Republic

The protein HydG together with the fusion protein HydEF seem to be indispensable for the maturation of
[Fe]-hydrogenase in the green alga Chalamydomonas reinhardtii . In addition, genes for all three proteins
were detected in all sequenced genomes of bacteria that also contain [Fe]-hydrogenase genes. We have
discovered HydG in the proteome of the hydrogenosomes, organelles of anaerobic ATP-production, in the
microaerophilic flagellate Trichomonas vaginalis, and have also found genes encoding homologues of HydE
and HydF in the T. vaginalis genome database. These results imply that the maturation process of [Fe]-
hydrogenase is conserved among eubacteria and eukaryotes. Phylogenetic analysis shows that HydG as
well as HydE in T. vaginalis and C. reinhardtii most likely share a common eubacterial ancestor, supporting a
monophyletic origin of [Fe]-hydrogenase maturases, and most probably also their target enzyme [Fe]-
hydrogenase.
REACTION OF ENTAMOEBA HISTOLYTICA TO DRUGS AND STRESS – A PROTEOMIC
APPROACH
               1           2                         1
David Leitsch , Iain Wilson , and Michael Duchêne
1
Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology,
                                            2
Kinderspitalg. 15, A-1095 Vienna, Austria; Department of Chemistry, Universität für Bodenkultur, Muthgasse
18, A-1190 Vienna

    The obligate anaerobic protist Entamoeba histolytica is a human gut parasite and can cause severe
abdominal and hepatic disease. Amebiasis annually affects several million people and claims up to 100000
human lives (WHO report on amebiasis, 1997). The gold standard chemotherapeutic agent is metronidazole,
a nitroimidazole derivative which has been in use for more than 30 years by now. Since metronidazole
treatment is very reliable and no clinically relevant resistant E. histolytica strains have been isolated so far,
only limited research addressing the cellular response of E.histolytica to this drug has been done. Evasion
mechanisms are conceivable because metronidazole is not damaging the cell unless being reduced by
ferredoxin. For Trichomonas vaginalis moderately metronidazole resistant strains have been isolated from
clinical specimens, and highly resistant strains were bred in the laboratory, making it in turn advisable to
shed some light on E.histolytica drug response.
      With the complete E.histolytica genome sequence at hand, analysis of protein expression by 2D PAGE
has been greatly facilitated in this organism. Protein extracts of challenged and unchallenged cells were
                                                                                         TM
compared and differentially expressed proteins identified with the help of the Melanie        proteomics software.
In addition to metronidazole, miltefosine (a drug shown to amebicidal) and physiological stress conditions
such as heat shock and osmotic stress were analyzed for their influence on E.histolytica protein expression.
By comparing the changes in the expression patterns, specific responses to metronidazole and miltefosine
can be defined. In case of metronidazole exposure the superoxide dismutase was observed at a shifted
position towards a more basic pI. In addition, several newly appearing spots were observed, which will be
subjected to mass spectrometry for identification.


This study was supported by grant P15960 from the Austrian Research Fund.
A NEW METHOD FOR CONTINUOUS CULTURE OF CRYPTOSPORIDIUM PARVUM

Mary Morada* and Nigel Yarlett
Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, New York, USA

Cryptosporidium parvum is an opportunistic pathogen of the intestinal tract of mammals. In common with
other members of the apicomplexa it undergoes a multistage life cycle which involves: a chemical and
environmental resistant oocyst stage, a motile sporozoites stage and reproductive merozoite stage. In vitro
                                                               2
culture of the parasite is typically performed using 75 cm tissue culture flasks containing a host-cell
monolayer in MEM containing 10% horse serum. Under these conditions the parasite undergoes
approximately two complete cycles of growth. The oocysts resulting from these cultures fail to infect new
flasks. Using a hollow fiber capillary bioreactor (HFB) we cultured a confluent layer of the human
adenocarcinoma cell-line (HCT-8) with continuos circulation of MEM containing 10% horse serum and the
following supplements: L-glutamine, HEPES, D-glucose, ascorbate, p-aminobenzoic acid, calcium
                                                      o
pantothenate, folic acid, penicillin-streptomycin at 37 C in a 5% CO2 incubator. The pH and glucose levels
were monitored daily, when the circulating glucose falls to 50% in 24 hours the system was infected with 1 x
  8
10 C. parvum oocysts. The oocysts excyst and infect the host cells which results in an initial increase in free
glucose, presumably due to destruction of the host cells. A population of the released merozoites form
oocysts and the host cell layer recovers which results in decrease of the free glucose. This cyclic change in
glucose persists as a damped oscillation for approximately 4 weeks, presumably due to loss of synchrony in
parasitemia. To date this culture has maintained the parasite for 3 months.



Funded by the National Institutes of Health AI49785 (NY).
COMPARATIVE EST ANALYSIS OF SYMBIOTIC ANAEROBIC PROTISTS OF TERMITE GUT
                   1,3                  1,3               2                   1,2                       1,3
Shigeharu MORIYA , Nemuri TODAKA , Kanako SAITA , Moriya OHKUMA                     and Toshiaki KUDO
1RIKEN Institute, 2Japan Science and Technology Agency (JST), 3Graduate School of Yokohama City
University

Although axenic cultivation has revealed thousands of microbial strains, recent progress in molecular
microbial ecology has shown that the vast majority of microorganisms in the eco-system still remain to be
determined. In this presentation, we propose an "environmental cDNA library" concept to understand a unit
of function expressed in an eco-system through the analyses of an "environmental cDNA library". For this
purpose, we constructed a cDNA library from protistan symbionts in the hindgut of various termites and
analyzed the contents.

Analyses of the DNA sequences in the 5'terminal region of approximately 1000 clones in the cDNA library
that had been clustered based on homology analysis by fasta. In all cases, a large portion of the clones
enabled assignment to the cellobiohydrolase (CBH) of the glycosyl hydrolase family 7 (GHF7). We also
found homologous sequences with GHF7-endogulucanase (EG), GHF5-CBH, GHF5-EG, GHF45-EG,
GHF10, 11 and 43-Xylanases. Interestingly, in almost all cases, same GHFs set was observed. Phylogenetic
analyses indicated that most of these sequences formed phylogenetically unique clusters in each GHF. The
GHF5 and xylanase sequences, however, that branched out from the clusters consisted of bacterial
enzymes. These phylogenetic relationships suggest horizontal transfers from bacterial origins.

The “environmental cDNA library” approach enables us to describe a rough figure for the expressed genes
within the symbiotic protistan community in the termite gut. The characterization of the transcribed genes as
described here are advantageous because genes in the environmental DNA represent mere potential activity
and they are not always expressed in the respective ecosystem.
KARYOTYPE AND THE COURSE OF MITOSIS IN GIARDIA INTESTINALIS.
              1                 1              2
Pavla Tumova , Eva Nohynkova and Jiri Kral
1                                      st                                                     2
Department of Tropical Medicine, 1          Faculty of Medicine, Studnickova 7, Prague 2; Department of
Genetics and Microbiology, Faculty of Science, Vinicna 5, Prague 2, Czech Republic.

An intriguing feature of a trophozoite of Giardia intestinalis is the presence of two nuclei, which are assumed
to be identical. In spite of significant advances in Giardia molecular biology, the nuclei have never been
karyotyped using conventional cytogenetic techniques and the accurate chromosome number is unknown.
Our cytogenetic analysis revealed that Giardia chromosomes are minute, without primary and secondary
constrictions. Surprisingly, in a trophozoite cell, karyotypes of the two nuclei differ in chromosomal number
and size. Moreover, odd chromosome(s) was present within a single nucleus. Therefore, our data contradict
with a simple diploid status of each nucleus, proposed by earlier studies. We propose that genome of each
nucleus displays aneuploidy which might lead to gradual haploidisation of its chromosomal set during
evolution. Mitosis of both nuclei proceeds asynchronously, with a distinct chromatid separation and
segregation into daughter nuclei. The revealed structural difference of Giardia nuclei implies inequality in
genetic information and might underlay a functional divergence of both nuclei, not yet postulated.
POSTERS
DOWN-REGULATION BY IRON OF THE CP65 CYSTEINE PROTEINASE INVOLVED IN THE
Trichomonas vaginalis CYTOTOXICITY TO HeLa CELL MONOLAYERS
                       a,b                b
Alvarez-Sánchez, ME          and Arroyo, R .
a
    Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, México, D. F., México.
b
    Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del I.P.N., Av.
IPN      #   2508,   Col.      San    Pedro    Zacatenco   CP   07360,     México,    D.F.,   México.    E-mail:
elizbethalvarezsanchez@yahoo.com.mx; rarroyo@cinvestav.mx.

Trichomonas vaginalis is an ancient protist responsible of trichomonosis, the most prevalent no viral sexually
transmitted disease in humans. T. vaginalis has high iron requirement for growth and multiplication and
regulates some virulence properties, such as adherence to host cells or resistance to complement lysis.
Several cysteine proteinases (CPs) have been involved in T. vaginalis virulence. One of them is a CP of 65
kDa, CP65, which participates in trichomonal cytotoxicity to HeLa cell monolayers. The aim of this study
was to test the effect of iron on the proteolytic activity of CP65 and parasite cytotoxicity. Iron responsiveness
of trichomonads grown under different iron concentrations was checked by addition of ferrous ammonium
sulfate (250 µM, high iron) to the culture medium (TYM-10% horse serum) or 150 µM 2,2´dipyridil to quelate
the iron (iron-depleted). After 24 h incubation only one duplication was observed in parasites grown in iron-
depleted medium, four duplications in regular culture medium (20 µM iron) and up to six duplications in high
iron (250 µM) conditions. The effect of iron on the levels of cytotoxicity of trichomonads grown in different
iron concentrations was tested on HeLa cell monolayers. Parasites grown in iron-depleted medium showed
2.17 times higher levels of cytotoxicity than those grown in high-iron medium.          Interestingly, the CP65
proteolytic activity analyzed by substrate gel electrophoresis of trichomonads grown in high-iron
concentrations had less CP65 proteolytic activity and lower levels of cytotoxicity than parasites grown in iron-
depleted medium.       Consistent with the effect of iron on the CP65 proteolytic activity the indirect
immunofluorescence assays with the anti-CP65 antibody showed a strong fluorescence of whole parasites
when trichomonads were grown in iron-depleted medium, while no fluorescence was detected on parasites
grown in high iron conditions. These data show that iron has a negative effect over the levels of cytotoxicity
of T. vaginalis, probably due to a reduction on the expression and proteolytic activity of CP65.
PROTEIN         PROFILE          OF     TRICHOMONAS              VAGINALIS       BY     TWO-DIMENSIONAL
POLYACRYLAMIDE GEL ELECTROPHORESIS
                                                             1
Juan Pablo Cifuentes-Ortiz, Javier Ambrosio-Hernández , Fernando Anaya-Velázquez and Felipe Padilla-
Vaca.

Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
                          1
Guanajuato, Gto. and          Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City,
México.

Trichomonas vaginalis is a protozoan which causes urogenital trichomoniasis. This infection is considered a
public health problem around the world, since it is one of the more common sexual transmitted diseases. In
order to cause tissue damage, this parasite attaches to host cells by using specific adhesion proteins.
Furthermore produces several potent proteases, mostly cystein proteases. In addition, more molecules are
also probably involved in its pathogenic mechanism. In order to know the protein pattern in GT-7 strain of T.
vaginalis, isolated in Guanajuato state, showing a low degree of virulence in vitro and in vivo, we
investigated by two-dimensional electrophoresis the profile of proteins constitutively expressed in cells
cultured in TYI-S-33 medium. Cellular fractions were derived from cell homogenates from log phase of
growth and were separated in the first dimension in an immobilized pH gradient and subsequently in the
second dimension by SDS-PAGE electrophoresis. A complex pattern of proteins was found, which was
reproducibly seen in independent experiments. The sequencing of these proteins is in progress and it will
allow us to start the proteomic analysis of protein patterns in strains of different degree of virulence, in order
to identify specific proteins present in virulent strains.
AN ARGININE-SPECIFIC MONO-ADP-RIBOSYL TRANSFERASE ACTIVITY IN ENTAMOEBA
HISTOLYTICA TROPHOZOITES

Eva E. Avila, Susana ML Fuentes, Mónica E. Silva, Araceli López, Angel H. Alvarez and Guadalupe
Martínez-Cadena

Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, P O
Box 187, Guanajuato, Gto, México. edilia@quijote.ugto.mx

Due to the important role of monoADP-ribosyl transferases (mADPRT) in physiological and pathological
events, we investigated whether the protozoan parasite Entamoeba histolytica had mADPRT activity.
Reactions were prepared using ameba free extracellular medium and intracellular fractions as source of
                                                                                32         +
enzyme and substrates for ADP-ribosylation. The source of ADP-ribose was { P}NAD , the proteins were
                                    32
analyzed by SDS-PAGE and the [ P]-labeled proteins were detected by autoradiography. In the crude
extracellular medium a main labeled 37 kDa substrate was secreted. The labeling of the 37 kDa substrate
                                                                                       +       32     +
was dependent on time, temperature, pH and the ratio between non-radioactive NAD and [ P]NAD in the
                                                               14                +
assay. The reaction was actually an ADP-ribosylation since [ C-carbonyl]NAD was not incorporated into
ameba substrates and a 75 fold molar excess of non-radioactive ADP-ribose did not compete in the
mADPRT reaction. One of the main ADP-ribosylated proteins in intracellular fractions was observed in a
                                                                         +
pellet sedimented at 160,000 g, in which the polymeric form of the NAD -dependent alcohol dehydrogenase
(EhADH2) was enriched. EhADH2 is among the metabolic enzymes absolutely required for amoeba growth.
We describe here the ADP-ribosylation of EhADH2, this modification did not have an effect on the enzymatic
activities, but it may regulate other functions of EhADH2. These results demonstrate the existence of at least
one mADPRT in E. histolytica. On the basis of sensitivity to NH2OH it is strongly suggested that both
extracellular and intracellular mADPRT from ameba are arginine specific enzyme(s). The physiological or
pathological relevance of ADP-ribosylation in Entamoeba histolytica remains to be determined.
DNA FROM TRICHOMONAS VAGINALIS INDUCES iNOS EXPRESSION IN MOUSE
MACROPHAGES

Patricia Cuéllar-Mata, E. Alderete-Ledezma, Martha Olivia Solís-Martínez and Sergio Arias-Negrete. Institute
for Research in Experimental Biology and School of Chemistry, University of Guanajuato. Noria Alta s/n, col.
Noria Alta, 36050, Guanajuato, Gto., Mexico.

Trichomonas vaginalis, a flagellated protozoan, infects the genitourinary tract of humans. Approximately
120-180 million people have trichomoniasis. This parasite damages host cells through a contact-dependent
mechanism. In lesions produced by this parasite, neutrophils, lymphocytes and macrophages have been
found. Moreover, specific anti-trichomonas antibodies have been detected. Macrophages have been
implicated in innate and adaptive immunity. Activated macrophages produce reactive oxygen intermediates
(ROI) and reactive nitrogen intermediates (RNI) which damage invading parasites. It is well established that
bacterial DNA, but not that from mammals, is mitogenic for B cells, and stimulates macrophages and
dendritic cells for NO synthesis. This stimulating activity relies on non-methylated CpG motifs. Little is known
about the role of parasitic DNA in natural and experimental infections. The purpose of this study was to
determine whether the DNA from Trichomonas vaginalis could induce iNOS and TNF-             mRNA expression,
iNOS protein synthesis and nitrite production in RAW264.7 mouse macrophages. DNA from T. vaginalis GT-
13 strain (a pathogenic strain) was utilized. Mouse macrophages were incubated with DNA, mouse
recombinant-IFN- and polymixin for 24 h at 37°C. iNOS mRNA expression (by RT-PCR) was 2.3 times
higher compared to non-stimulated cells. TNF-       mRNA expression (by RT-PCR) was not modified under
parasite DNA stimulation. NO production was measured as nitrite production (determined by the Griess
reaction). Macrophages synthesized 23 times more nitrites than control after trichomonas DNA stimulation.
iNOS protein was detected by western blot analysis using specific anti-iNOS antibodies. Interestingly, for
iNOS protein detection and nitrite synthesis, it was not necessary to add rIFN- . Polymixin inhibited iNOS
protein synthesis by 30%. Our results indicate that DNA from T. vaginalis was capable of stimulating mouse
macrophages, possibly through hypo-methylated CpG sequences. This is a possible explanation for the
immune effector mechanism during the invasive process in humans.



Project supported by CONACYT, Mexico.
DETECTION AND GENOTYPING OF GIARDIA DUODENALIS FROM HUMAN AND ANIMAL
ISOLATES IN ITALY

D. Di Cave1,2

1 Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di Roma “Tor Vergata”, Rome, Italy. 2
Azienda Ospedaliera Universitaria Policlinico “Tor Vergata”, Rome, Italy.

The aim of the study was to investigate the presence of Giardia duodenalis in samples from humans,
domestic and farm animals in Italy and to genotype the isolates. Few data on the prevalence, animal and
public health impact of Giardia isolates in Italy are available. Moreover, the diagnosis is mainly performed
using conventional microscopy, and little is known regarding the genetic characterisation of isolates. In order
to address these questions, faecal samples from symptomatic patients were obtained from several
laboratories. The study population comprised 24 males and 35 females, ranging in age from 0 to 63 years.
To   determine    the   presence   of   Giardia,   faeces   were   examined   by   direct, concentration   and
immunofluorescence methods and by PCR. In addition, stool samples from 113 dogs, 10 calves and 1 cat
were analysed microscopically. The majority of dog samples and the only one tested cat originated from
kennels, all the calf specimens originated from dairy farms.

DNA was extracted directly from faeces using commercial kit. Isolate genotyping was carried out using a
nested PCR, and a 200-bp region of the 5’ end of the small subunit (16S) rRNA gene was amplified and
sequenced. Relating to clinical isolates, 19 faecal samples were recovered as positive microscopically, 40
were negative. Amplification by PCR confirmed the positive samples whereas 8 new cases were assessed.
Sequence analysis confirmed all 27 isolates as G. duodenalis corresponding both human Assemblages A
(17) and B (9) and one mixed Assemblage. Although laboratory diagnosis is made conventionally by
microscopic examination of stool samples, the high levels of Giardia PCR positives show that PCR-based
methods are more sensitive and specific for the detection of low numbers of cysts with respect to sub-clinical
levels of Giardia infection.

Concerning animal samples, a total of 21 Giardia isolates were genetically characterised. Among dogs,
76.5% of isolates showed the dog-specific genotypes (Assemblages C, D and C/D mixed Assemblage) and
23.5% exhibit potential zoonotic genotypes (Assemblage A and A/C mixed Assemblages). The cat isolate
belonged to Assemblage A, whereas the sequences among the isolates from calves were found to
correspond to hoofed-livestock genotype Assemblage E. These data suggest that infection of humans by
zoonotic genotypes from domestic animals could be of low epidemiological significance, although possible.
CHARACTERISATION OF A DYNAMIN LIKE PROTEIN IN GIARDIA: GiDLP IS INVOLVED IN
CYST FORMATION AND ENDOCYTOSIS

Verena Gaechter and Adrian B. Hehl

Institute of Parasitology, Winterthurerstr. 266a, CH-8057 Zürich

Dynamin and dynamin-like proteins (DLPs) are conserved large GTP-binding mechanoenzymes, which play
an important role in a wide range of cellular processes, including meiotic spindle pole separation,
mitochondrial fusion and division, vesicle scission, viral resistance, protein trafficking and various forms of
endocytosis. We report here the characterization of the dynamin-like protein (GiDLP) in the early diverged
eukaryote Giardia lamblia. GiDLP was shown to be associated to the lysosome-like peripheral vesicles in
trophozoites, where it co-localizes extensively with giardial clathrin. However, during encystation, GiDLP is
recruited to the Golgi-like encystation-specific vesicles (ESVs) and associates with the cytoplasmic side of
the ESV membrane. The function of GiDLP in the encystation process was examined by conditional
expression of a dominant-negative mutant (K43E) GiDLP variant. The single amino acid change abolished
binding to ESV membranes of the K43E            mutant but did not inhibit ESV formation. However, cyst
development was reduced by 90 - 98% compared with wild type and GiDLP over-expressing cells,
suggesting an essential function of GiDLP in ESV maturation or secretion.

Dynamin and DLPs are also involved in endocytic processes. To evaluate the effect of a dominant-negative
GiDLP on endocytosis we compared uptake of biotinylated surface proteins by wild-type and transgenic
trophozoites. Immuofluorescence experiments show that wild type cells endocytose biotinylated surface
proteins very efficiently while mutants show a highly reduced biotin uptake. Quantitative analysis to define
the contribution of GiDLP to endocytosis is under way.

Our results suggest that GiDLP is a multifunctional enzyme involved in exocytic and endocytic processes in
Giardia. Current investigations aim to clarify the exact role of GiDLP, especially its interaction with other
proteins such as clathrin.
HUMAN sIgA DEGRADATION BY SURFACE PROTEASES OF Entamoeba histolytica.

Rosa María García-Nieto, Sergio Arias-Negrete and Eva E. Avila. Instituto de Investigación en Biología
Experimental, Facultad de Química, Universidad de Guanajuato, Noria Alta S/N, Col. Noria Alta, 36050,
Guanajuato Gto., nietor@quijote.ugto.mx.

Secretory IgA plays a crucial role in the host immune response as a first line of defense against pathogens
which colonize and invade surfaces bathed by secretions. There are two subclases of human IgA, IgA1 and
IgA2, and they play key roles in protecting the mucous membranes from attack by pathogenic
microorganisms and their products. A few bacteria produce enzymes called IgA1 proteases capable of
cleaving only IgA1 and not IgA2. They cleave in the hinge region and release intact Fab and Fc (or Fc.SC).
IgA1 has 22 amino acids in the hinge region, IgA2 has only 9 en this region, that is the reason because of
the difference of the susceptibility to the proteases. Entamoeba histolytica is the causative agent of
amoebiasis. Among the virulence factors are more than 20 cysteine protease. These proteases can cleave
the human sIgA to evade the immune response.             This degradation has been reported but is not
characterized functionally, it is not known if after this process the sIgA is capable to bind and agglutinate
Entamoeba histolytica trophozoites. Serum and secretory IgA were exposed to amoebic protease of the
surface of fixed trophozoites. Both types of IgA were degradated by surface amoebic protease. Degradation
was faster in the IgA from serum. It has been reported that secretory component protects sIgA for protelytic
degradation. For that reason it is important to test secretory component resistance to amoebic proteases.
We observe degradation of both free and IgA-associate secretory component. Degradated antibodies were
capable to agglutinate live Entamoeba histolytica trophozoites. In our conditions it was observed that serum
or secretory IgA were functional after degradation.
EFFECTS         OF       POLYAMINE          DEPLETION       BY    DFMO       ON   HYDROGENOSOMES             IN
TRICHOMONAS VAGINALIS
                     1,2,3              2                  1                 1                  1,2          3
Kristina M. Harris       , Burt Goldberg , Michael Turner , Anthony Hayes , Katey M. Lemar , Nigel Yarlett ,
           1
David Lloyd .
1
Microbiology (Bios1), Cardiff University, Park Place, Cardiff CF10 3TL, Wales, UK.
2
Department of Chemistry, New York University, Silver Center, New York, NY 10003, USA.
3
Haskins Laboratories and Department of Chemistry and Physical Sciences, Pace University, 41 Park Row,
New York, NY 10038, USA.

The correlation between polyamine metabolism and the hydrogenosomes in Trichomonas vaginalis was
examined using three different methods. When examined by fluorescence microscopy, morphological clarity
of the hydrogenosomes was lost after T. vaginalis was exposed overnight to 5mM difluoromethylornithine
(DFMO); this compound arrests polyamine synthesis by inhibiting the enzyme ornithine decarboxylase
(ODC). When examining samples prepared for transmission electron microscopy, samples grown overnight
in 5mM DFMO were seen to have electron-dense inclusions surrounding, and inside, the hydrogenosomes.
Using flow cytometry, a decrease in fluorescence of polyamine-depleted cultures stained with 0.50nM
Dio(C6)2 or 0.50nM TMRE was observed. Thus, since fluorophore uptake correlates with the trans-
membrane       electrochemical    potentials   of   the   organism,   the   combined   plasma    membrane   and
hydrogenosomal membrane potentials decreased after arresting polyamine synthesis. Further, upon
exposing these polyamine-depleted cells to 5mM spermine or 5mM spermidine for 7h prior to using the
same flow cytometry protocol, the fluorescence (and therefore plasma membrane and hydrogenosomal
membrane potential) was found to recover. We can conclude that the depletion of polyamines using DFMO
disturbs hydrogenosomal structure and function. The presence of polyamines is essential in T. vaginalis in
order to maintain the normal, fully-functional hydrogenosome, as well as a normal plasma membrane and
hydrogenosomal membrane potential.
TRICHODB: A WEB-BASED RESOURCE FOR THE TRICHOMONAS VAGINALIS GENOME
                   1                 2                    3                      3                    1
Richard D. Hayes ; Jane M. Carlton ; Michael J. Cipriano ; Andrew G. McArthur ; Patricia J. Johnson
1
    University of California – Los Angeles, CA, USA
2
    The Institute for Genomic Research, Rockland, MD, USA
3
    Marine Biological Laboratory, Woods Hole, MA, USA

Trichomonas vaginalis is one of the most prevalent non-viral sexually transmitted infectious parasitic protists
in the world, with over 200 million new cases of infection every year. Sequencing of a laboratory strain of T.
vaginalis was completed in April 2005 by the Institute for Genomic Research. Genome curation and the
development of an accurate and complete annotation is an important next step in the study of this protist. To
facilitate involvement of the T. vaginalis research community in this ongoing process, a web-based database
application has been developed (available at http://www.trichodb.org/). Based primarily on the Generic
Model Organism Database schema implementation for Giardia lamblia produced at the Marine Biological
Laboratory, this website will include features expected of current genome websites such as full genomic data
graphical browsing. A password protected system of individual researcher accounts will house individual
contributions to the genome annotation from a wide range of potential experimental sources in a secure
environment, simultaneously providing an easy method for transition of annotation data to the public
database upon publication.
GENE EXPRESSION OF ENTAMOEBA HISTOLYTICA UNDER THE INFLUENCE OF
METRONIDAZOLE AND ALKYLPHOSPHOCHOLINES
            1                      2                  2                        1
Margit Hofer , Evelyn Hatzenbichler , Martin Schreiber , and Michael Duchêne
1
Department of Specific Prophylaxis and Tropical Medicine, Center for Physiology and Pathophysiology,
2
Department of Obstetrics and Gynecology, Medical University Vienna, Austria

    Our aim is to use oligonucleotide microarrays to analyze the effects of alkylphosphocholines and
metronidazole on gene expression of Entamoeba histolytica. We generated annotations by searching for
homologs of known enzyme-encoding genes using the web version of the Roche "Biochemical Pathways".
The known sequences from reference species, mostly human and E. coli, were blasted against TIGR and
Sanger contigs and E. histolytica homologs were entered in a database. In addition, two databases of
cytoskeletal genes and stress genes were generated.

    Our microarray includes genes encoding glycolysis enzymes, cytoskeleton components, surface
antigens, lipid metabolism enzymes and stress-related proteins. Remarkably, many important house keeping
genes, e.g. glyceraldehyde phosphate dehydrogenase (GAPDH), were only found in one copy in the
genome, whereas cytoskeleton genes such as actin were found in large gene families, so we selected
subsets based on multiple alignments of these families. In total, we compiled 221 oligonucleotides to be
printed on our array. They were synthesized with 5´-amino modifications and spotted to expoxy-modified
slides.

    We prepared total RNA from E. histolytica cells which are treated with different concentrations of
metronidazole as probe and use total RNA of untreated cells as control. The transcribed cDNA is labeled
with Cy5 (probe) and Cy3 (control) respectively. Hybridization is performed in 5xSSC, 30% formamide at
37°C for 16 hours. The slides are washed in decreasing concentrations of SSC and scanned in a GenePix
4000A scanner (Axon Instruments) at 10-µm resolution.

    Preliminary results will be presented in September, up to now no great differences in gene expression
between cells treated with metronidazole and untreated cells have been observed.



This work was supported by grant P15960 from the Austrian Science Fund.
GENETIC DIFFERENCE OF E. HISTOLYTICA BY SUBTRACTIVE HYBRIDIZATION AND
DIFFERENTIAL GENE EXPRESSION

M. Pilar Crisóstomo-Vázquez, Enedina Jiménez-Cardoso

Laboratorio de Investigación en Parasitología, Hospital Infantil de México Federico Gómez, México, D. F.,
México

The mayor frecuence of E. histolytica in human remain asyntomatic and a small portion of infections E.
histolytica penetrate the intestin and is capable disseminate to the organs and the development liver
abscesses. In order to understand this differences we are using substractive libraries to identify genes that
are differentially expressed in this two kinds of trophozoites.

Objetive: Compared the cDNA expression between E. histolytica isolated from liver abscesses of infected
hamsters and those grown under normal culture conditions.

Material and Methods: The cDNA synthesis from mRNA(+) was obtain of trophozoites isolated from liver
abscesses “A” and those grow in axenic culture “B”. n order to have a first screening the made the
hibridazion cDNa (A) with cDNA B. The elute double stranded fraction was using Differential Display
polymerase chain reaction (DD-PCR) which arbitrary primers of 14-nucleotides (nt) using a “care” oligo dt
and AT, CG, GC (master primer) the conditions of PCR was allowed the restriction of low annealing
temperatures to the first PCR cycle only. 0ach arbitrary primer produced 10 amplified cDNA fragments.

Results: The differences of DD.PCR between abscess “A” and “B” revealed differences in cDNA from
abscess-derivate amoebae “A”. After excision from the bot and amplification was clones and sequences nine
sequences of 233-432 bp. BLAST searches in TIGR Entamoeba genome shows proteins like Ubiquitin,
ciclophylin, ferredoxin among others.

Conclutions: We found differences in some amplicons specific for abscess-derivative amoebae that we don’t
see in axenic cultures.
IDENTIFICATION OF NOVEL REGULATORY MECHANISMS OF THE MULTIFUNCTIONAL 14-
3-3 PROTEIN IN GIARDIA DUODENALIS


           a                      b                      b                a
Marco Lalle , Anna Maria Salzano , Marco Crescenzi and Edoardo Pozio
a                                                                         b
Department of Infectious, Parasitic and Immunomediated Diseases, Department of Environment and
Primary Prevention, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161 Rome, Italy. E-mail:
marco.lalle@iss.it

The multifunctional 14-3-3s are a family of highly conserved eukaryotic proteins able to bind different target
proteins containing specific motifs usually phosphorylated on serine or threonine. The involvement of 14-3-3s
in many cellular processes, such as cell cycle, differentiation, apoptosis and signal transduction pathways,
has been documented in different organisms but limited information are available concerning their role in
parasites. We have chosen the flagellated protozoa parasite Giardia duodenalis (syn. lamblia or intestinalis)
as a model to further investigate on 14-3-3 functions.
By an in silico screening of the complete G. duodenalis genome (strain WB-C6), we confirmed the presence
of a single 14-3-3 homologous gene. The predicted 248 amino acid protein, named g14-3-3, showed an high
degree of identity (60%) with the epsilon group of 14-3-3s. The g14-3-3 coding sequence has been cloned
and expressed in bacteria as GST-fusion protein and, by overlay experiments, both the dimerization property
and the interaction with ligands containing phosphorylation-dependent and -independent binding motifs have
been demonstrated for the recombinant g14-3-3. Using a polyclonal anti-peptide antibody, raised against a
unique 18 amino acid sequence at g14-3-3 N-terminus, the expression profile and the intracellular
localisation of the protein have been studied throughout the different parasite life stages, together with the
transcription level of the mRNA. An affinity cromathography has been set up to purified the native g14-3-3
from total soluble proteins obtained from G. duodenalis trophozoites and encysting cells. The purified g14-3-
3 has been extensively analyzed by MALDI-MS leading to the identification of multiple post-translational
modifications (PTM). The putative regulatory function of these PTM is under investigation.
IDENTIFICATION AND LOCALIZATION OF THE CYSTEINE PROTEINASE TvCP12 OF
Trichomonas vaginalis USING POLYCLONAL ANTIBODIES AGAINST A SYNTHETIC
PEPTIDE FROM THE MOST DIVERGENT REGION OF THIS PROTEIN

León-Sicairos, C. R. and Arroyo, R.

Departamento de Patología Experimental. Centro de Investigación y de Estudios Avanzados del IPN
(CINVESTAV-IPN). Av. I.P.N. # 2508, Col. San Pedro Zacatenco. CP 07360, México. D.F., México. Email:
claudia_leonsicairos@yahoo.com.mx; rarroyo@cinvestav.mx.

Trichomonas vaginalis is the causative agent of trichomonosis one of the most common sexually transmitted
diseases in humans. This protozoan has multiple proteinases that are mainly of the cysteine (CP) type,
some of which are known to be involved in the parasite virulence.                 We previously identified and
characterized a novel T. vaginalis CP-encoding gene, tvcp12, which is another gene that is negatively
regulated by iron. Tvcp12 is 948 bp long and encodes a predicted 34.4 kDa precursor protein that has the
characteristics of the papain-like CP family. However, the role that this proteinase plays in T. vaginalis is still
unknown. In this work we designed a synthetic peptide from tvcp12 deduced amino acid sequence. By
using the database of the T. vaginalis Genome Project (http://www.tigr.org/) we search for the most divergent
region from tvcp12 as compared with the other papain-like CP sequences. Based on the Kyte-Doolittle and
Hopp-Woods scale (http://workbench.sdsc.edu/), we looked for the predicted potentially antigenic and
hydrophilic regions of tvcp12 sequence.         3-D theoretical reconstruction of TvCP12 deduced protein
sequence (http://expasy.ch/) using the crystal structure data of papain-like CPs with high homology to
TvCP12 showed that the selected decapeptide DVSKGYAKVT (at position 200-210 aa residues)
corresponds to an accessible region in the molecule surface. The synthetic peptide was coupled to 8-MAP
and used to immunize five male Balb-c mice to produce specific anti-TvCP12 antibodies. Dot-Blot assays
showed that the anti-TvCP12 antiserum reacted with the synthetic peptide and with trichomonad extracts.
Western blot, immunoprecipitation, and immunocytochemical assays with the polyclonal antibodies to
TvCP12 synthetic peptide helped us to identify and localize this proteinase among the CPs from the 30-kDa
region of T. vaginalis.
INFLUENCE OF TRICHINELLA SPIRALIS-INDUCED INTESTINAL INFLAMMATION ON A
GIARDIA LAMBLIA INFECTION IN THE MURINE HOST
               1                    1                 1                 2                 1                     2
Norbert Müller , Nicole von Allmen , Selina Christen , Ursula Forster ; Bruno Gottstein , and Monika Welle
                         1                            2
Institutes of Parasitology and Veterinary Pathology , University of Berne, Berne, Switzerland

Giardia lamblia is a common intestinal dwelling protozoan and causes diarrhoea in humans and animals
worldwide. In the past, various immunological and non-immunological parameters of a G. lamblia infection
have been determined particularly in experimental hosts. However, only very few of these investigations took
into account that the outcome of giardiasis may be influenced by other infections that had eventually
occurred prior to a G. lamblia infection. In this context, we performed a study to assess in the mouse model
the impact of a nematode (Trichinella spiralis) infection on the course of a subsequent G. lamblia infection.
For the infection experiments, we used the well-characterized G. lamblia clone GS/M-83-H7. In our study,
the intestinal phase of the T. spiralis infection coincided with intestinal inflammation and was associated with
a striking local mast cell activation. In T. spiralis/G. lamblia-double-infected animals, the transient
inflammatory reactions generated an intestinal environment which strongly promoted growth of G. lamblia
trophozoites. This elevated G. lamblia infection intensity strikingly correlated with intestinal mast cell
infiltration and IgE-dependent mast cell degranulation. Interestingly, a G. lamblia single-infection also
resulted in intestinal mast cell accumulation but degranulation was found to be triggered in the absence of
IgE. Recently, Li et al. (Infection and Immunity 72, 6642-6649 [2004]) provided evidence that mast cells are
involved in resolution of a G. lamblia infection in mice. An overall evaluation of the present and previous
findings as outlined above indicates that intestinal mast cells consist of distinct subsets that either promote,
or inhibit, intestinal growth of G. lamblia trophozoites in mice. It is evident that in natural hosts, a G. lamblia
infection often occurs in a situation where the intestinal tissue is inflamed as consequence of a parasitic,
bacterial and/or viral infection. Our data demonstrate that a local inflammation induced by a nematode
infection favours establishment and maintenance of a G. lamblia population within the murine intestine. The
process of inflammation has been found to influence not only the immunological, but also the physiological
conditions inside the intestinal environment. Accordingly, approaches investigating giardial growth in relation
to an eventual intestinal inflammatory reaction will provide novel information on the immunological and
physiological functions that promote either resistance or susceptibility to a G. lamblia infection.
CRYPTOSPORIDIOSIS CIRCULATION IN DIFFERENT REPRESENTATIVE SAMPLES FROM
MAPUTO PROVINCE, MOZAMBIQUE

Emilia Noormahomed*, Noémia Nhancupe*, Catherine Kauffmann-Lacroix**, Carmen Mascaró***, Mauro M.
Colombo****
* Parasitology Sector, Department of Microbiology, Faculty of Medicine, Eduardo Mondlane University, PO
Box 257 Maputo- Mozambique, e-mail: eraul@teledata.mz.
** Laboratoire de Parasitologie et Mycologie Médicale, Centre Hospitalo-Universitaire de Poitiers, France.
***
      Departament of Parasitology, Institute of Biotechnology, University of Granada, Spain.
****Dept. of Cellular Developmental Biology, University of Rome, La Sapienza, Italy.

Crytosporidium sp., an opportunistic anaerobic protozoon, is a major cause of morbidity and mortality in
imunocompetent children and imunocompromised patients throughout the world, mainly in developing
countries, in association with deficient conditions of personal and environmental hygiene.
In Mozambique, no recent systemized data are available on cryptosporidiosis, either in children or in adult
general population.
A total of 440 stool samples from different social groups were studied: 269 from healthy children aging from
0 to 15 years, 75 from AIDS patients aging from 17 to 49 years and 96 from neuropsychiatric patients, aging
from 8 to 58 years; all were living in the urban environs of Maputo, Mozambique.
The aim of the study was to ascertain the prevalence of cryptosporidiosis, and to analyze the association of
different socio-economic as well as environmental risk factors, with circulation dynamics of the parasite in the
three groups.
The overall prevalence of Cryptosporidium sp. was 9.33% in the asymptomatic children group, 52.90% in
AIDS patients (with and without diarrhea) and 54.95% in patients with neuropsychiatric disturbance.
Cryptosporidium prevalence in asymptomatic children and in AIDS patients is almost consistent with other
studies data, at least in developing countries. Surprisingly the neuropsychiatric patient group, not specifically
included in the study for gastrointestinal symptoms, showed a very high prevalence of cryptosporidiosis: this
might be due to poor healthy and living conditions, directly related to their psychiatric behavior.
In Mozambique, according to health system guidelines, unless specifically requested, laboratories do not
specifically diagnose Cryptosporidium presence and the organism cannot be detected using ordinarily
procedures for parasite detection. This study revealed that stool samples should be specifically examined for
Cryptosporidium sp. oocysts, since it widely circulates in Maputo province population. It also shows that
healthy carriers constitute a serious problem for public health, and may represent a risk for
immunodepressed patients by oro-faecal transmission through food and water contamination.
ANALYSIS BY RANDOM AMPLIFIED POLYMORPHIC DNA OF CLOSELY RELATED
TRICHOMONAS VAGINALIS STRAINS

Felipe Padilla-Vaca, Ana Estela Gamiño-Arroyo, Minerva Paola Barrios-Ceballos and Fernando Anaya-
Velázquez.
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato,
Guanajuato, Gto., México.

Trichomonas vaginalis is the causative agent for human trichomoniasis. This infection is the most common
non-viral sexually transmitted disease and is considered a public health problem around the world. The
infection in women may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high
prevalence, the genetic variability, virulence and geographic origin of this organism are poorly understood.
To address these issues, the random amplified polymorphic DNA (RAPD) technique was used to determine
genetic differences among 12 isolates of T. vaginalis from Guanajuato state and 2 reference strains from
ATCC (C-1:NIH and RFC-1) using 12 different random primers. Our results showed genotypic similarities
among all the isolates from Guanajuato and a clear different genotypic profile with respect to the C-1:NIH
strain. A modified RAPD protocol was implemented to find a single or very few mutational events in the no
polymorphic DNA isolates. No concordance was found between RAPD patterns of T. vaginalis strains and
their virulence.
INTERNAL SIGNAL-DEPENDENT TARGETING OF GIARDIA ISCS INTO TRICHOMONAD
HYDROGENOSOMES

Petr Rada, Pavel Dolezal, and Jan Tachezy
Department of Parazitology, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic

Proteins directed into the mitochondria are synthesized in cytosol with either an N-terminal or internal
targeting signals. Both types of targeting information are recognized by the outer (TOM) and inner (TIM)
membrane translocases. The mitochondrial matrix proteins are further translocated through the TIM23
complex, with energy supplied by a PAM complex. After translocation, N-terminal presequences are cleaved
by a matrix-located processing peptidase. Similar mechanisms mediate protein targeting into mitochondria-
related organelles such as hydrogenosomes and mitosomes. Our initial work on the proteins assembling Fe-
S clusters in Giardia intestinalis showed that two mitosomal proteins, GiiscU and [2Fe2S] ferredoxin, have
predicted N-terminal extensions, while such an extension was absent in GiiscS. Comparison of GiiscS with
mitochondrial IscS homologs possessing targeting presequences revealed the presence of two unique
insertions in GiiscS. The insertion located closer to N-terminus is rich in glutamic acid, while the distal one
contains polar and positively charged amino acids. To test possible involvement of these insertions in GiiscS
translocation, the insertions were deleted and translocation of modified GiiscS into hydrogenosomes of
Trichomonas vaginalis was studied. Deletion of internal sequences completely inhibited translocation of
GiiscS into the organelles and the protein accumulated in cytosol. These results suggest that these unique
insertions of GiiscS may represent internal signals for the protein targeting and translocation.
STRUCTURAL         AND     FUNCTIONAL         CHARACTERIZATION            OF    THE    EHRABB        GENE
PROMOTER OF ENTAMOEBA HISTOLYTICA
                     1                  2               1                         1
Mónica Romero-Díaz , Consuelo Gómez , Esther Orozco , and Mario A. Rodríguez .
1
Departamento de Patología Experimental. Centro de Investigación y de Estudios Avanzados del IPN. A.P.
                                        2
14-740, México, D.F. 07000, México. Programa Institucional de Biomedicina Molecular, ENMyH-IPN,
Guillermo Massieu Helguera No. 239. Fracc. La Escalera. Ticomán, CP 07320, México, D.F., México.

EhrabB is an Entamoeba histolytica gene encoding a Rab GTPase involved in phagocytosis. This gene is
situated at 332 bp upstream, but in the complementary strand, of the gene encoding the EhCP112
polypeptide, also involved in E. histolytica pathogenesis. This genomic association suggests that
transcription regulatory elements of both genes may be shared or overlapped and, therefore, they may have
a coordinated expression regulation. To elucidate the molecular mechanisms controlling the EhrabB gene
expression, here we determined its transcription start site and carried out the structural and functional
analysis of the regulatory region responsible for its expression. The search into 850 bp upstream of the
EhrabB start codon revealed the absence in this segment of E. histolytica consensus sequences for Inr,
GAAC element and TATA-box. This region neither contains the upstream regulatory elements (URE) that
control the Ehgl5 gene transcription. In contrast, the EhrabB promoter region contains DNA consensus
sequences for the binding of well-known eukaryotic transcriptional factors, like C/EBP, CdxA, GATA-1 and
heat shock factor (HSF). Primer extension analysis revealed several transcription start sites for EhrabB, as
often found in TATA-less promoters. The shortest EhrabB transcript initiates at 72 bp upstream of its start
codon and the residue located in this point was arbitrarily designed as position +1. We cloned into the
promoter-less vector pBS-CAT-ACT a DNA fragment comprising nucleotides from             -683 to +96, which
includes three heat shock response elements (HSE). Trophozoites transfected with this construction showed
higher CAT activity under heat shock than at 37 °C, suggesting that HSE sequences could be a functional
role on the EhrabB gene transcription. Then, we analyzed the ability of different fragments from the 5’
flanking region of EhrabB gene to drive the expression of the cat reporter gene on transfected trophozoites.
Results showed that: i) DNA sequences located between positions -428 to -683 negatively control the
EhrabB transcription; and ii) sequences situated between positions -257 to -428 activate the EhrabB gene
transcription. The DNA segments that stimulate and diminish the EhrabB gene transcription are inside the
open reading frame of the Ehcp112 gene, confirming that expression of both genes may be coordinately
regulated.
IDENTIFICATION AND LOCALIZATION OF TVLEGU-1, ONE OF THE LEGUMAIN-LIKE
CYSTEINE PROTEINASES OF Trichomonas vaginalis

Salas-Garrido C G*, Ortega-López J **, Arroyo R *

*Departamento de Patología Experimental **Departamento de Biotecnología y Bioingeniería, Centro de
Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN). Av. IPN #2508, Col. San Pedro
Zacatenco, CP 07360. México, D.F. México. E-mail: gerardito@lycos.com; rarroyo@cinvestav.mx

Trichomonosis is one of the most common sexually transmitted disease worldwide caused by Trichomonas
vaginalis.   Cysteine proteinases (CPs) are abundant in T. vaginalis and most of them belong to the
cathepsin-L subfamily of clan CA. Recently, we identified by 2-D substrate gel electrophoresis of parasite
extracts the presence of at least three legumain-like proteinases in the 30 kDa region. Two of the legumain-
like genes Tvlegu-1 and Tvlegu-2 were cloned, sequenced, and grouped with CPs of the aspariginyl
endopeptidase subfamily of clan CD. To identify, characterize, and immunolocalize this CP in T. vaginalis by
specific polyclonal antibodies a specific peptide from TVLEGU-1 sequence was synthesized. To select the
most divergent peptide from all the legumain-like genes the database of the T. vaginalis Genome Project
(http://www.tigr.org/) was searched. Thus, ten legumain-like encoding genes were identified and analyzed
by multiple alignments with the CLUSTAL-W program. The most divergent region of TVLEGU-1 was found
at the carboxy terminus between amino acid (aa) residues 258-388. Based on the analysis of TVLEGU-1 by
the Hopp-Woods scale for hydropathy and antigenicity, the dodecapeptide NEVVAPKADAKI (at position
206-271 aa residues) was synthesized. In addition, the 3-D theoretical reconstruction model obtained with
the LOOPP V3.00 program, predicted that this peptide is exposed on the surface of TVLEGU-1 3-D model.
The synthetic peptide was coupled to 8-MAP and five male Balb/c mice were immunized to produce
antibodies. By Dot-blot the anti-TVLEGU-1 antiserum reacted with the synthetic peptide and trichomonad
extracts. Western blot, immunoprecipitation, and immunocytochemical assays allowed us to identifiy and
localize TVLEGU-1 among the CPs of the 30 kDa region in T. vaginalis extracts.
THE SECOND INHIBITOR OF CYSTEINE PROTEASES FROM ENTAMOEBA HISTOLYTICA,
AMOEBIASIN-2, MAY FUNCTION EXTRACELLULARLY.

Mirela Šarić, Anke Harsman, Sabine Riekenberg, Henning Scholze
University of Osnabrueck, Dept. Biology/Chemistry,

Cysteine proteases are essential pathogenicity factors of the protozoan parasite Entamoeba histolytica. They
are responsible for the penetration of the human gut wall and for establishing abscesses in internal organs.
In view of this, cysteine proteases would be logical targets for new chemotherapeutics. This concept is
supported by the fact that trophozoites treated with cysteine protease inhibitors exhibit significantly reduced
virulence. Endogenous cysteine protease inhibitors have been found in various parasitic protozoa. Among
these, the chagasins, the first of which has been discovered in Trypanosoma cruzi, seem to be widespread.
Recently, by screening the data base of the E. histolytica genome project of the Sanger Institute, we
identified two chagasin homologues in E. histolytica, which we named amoebiasin-1 and amoebiasin-2 and
demonstrated its presence in trophozoites. Whereas recombinant amoebiasin-1 effectively inhibited the
peptidase activity of both papain and a soluble trophozoite extract, amoebiasin-2 does not inhibit papain, but
acts against a trophozoite extract as well as cathepsin B from human liver. The overall sequence identity
between both amebic inhibitors at 27% is relatively low suggesting that both genes are not a result of a gene
duplication. In contrast to that of amoebiasin-1, only the predicted amino acid sequence of amoebiasin-2
contains a typical signal sequence indicating that the protein may be extruded out of the cell. Both proteins
are distinguished by three conserved regions: LTE, NPS/TTGYS/AW and IXLXYXRPW. Preliminary
ELISA/Western blot studies indeed suggest that amoebiasin-2 is actually secreted by the trophozoites. To
test whether some of these motives are actually involved in inhibition, we synthesized the heptapeptides
GNPTTGF and GYSRPWE and tested them for their inhibitory activity towards papain and towards a soluble
trophozoite extract. In these experiments, only the peptide GNPTTGF inhibited the proteolytic activity of
papain and that of the trophozoite extract. Thus, NPPTG-based peptides may be seen as starting point for
the rational design of an anti-amoebic drug.
Although both amoebiasins inhibit amoebal cysteine proteases, the exact functions of these proteins - in
housekeeping or within the host-parasite-interaction - are unknown. The lack of a signal sequence suggests
that trophozoites utilise amoebiasin-1 as protector against endogenous cysteine proteases, whereas
amoebiasin-2 having a signal sequence may act extracellularly against host proteases. In the latter case,
amoebiasin-2 is directly involved in the host/parasite-interaction and might constitute an important
pathogenicity factor.
TRANSCRIPTONAL RESPONSE OF TRICHOMONAS VAGINALIS TO DIFFERENT IRON
SOURCE BY cDNA MICROARRAY

Jyh-wei, Shin and Petrus Tang

Department of Parasitology, Core Laboratory of Microarray, College of Medicine, National Cheng Kung
University, Tainan, Taiwan and Molecular Regulation & Bioinformatics Laboratory, College of Medicine.
Chang Gung University, Taoyuan, Taiwan.

As the Trichomonas vaginalis G3 genome sequencing project nears completion, functional analyses that
provide a global genetic perspective on biological processes are important. To provide a better
understanding on the gene expression reportorial of T. vaginalis, we constructed a cDNA array chip
(TvARRAY.V1) from expressed sequence tag (EST) clones. Around 26,000 ESTs were clustered into 3896
contigs and 5125 singletons. cDNA inserts from 7676 non-redundant EST clones were amplified by PCR
and arrayed onto the TvARRAY chip. Microarrays were screened with total RNA isolated from mid-log phase
cells grow in YIS medium supplemented with either ferrous iron or hemoglobin. We showed that 9.6% and
11.3% of the genes were upregulated and downregulated respectively with at least two-fold in response to
hemoglobin treatment. Around 289 transcripts related to energy metabolism, cytoskeleton, and signal
transduction showed at least five-fold differences. Based on the Gene Ontology classification, the
upregulated and downregulated genes covered 11 and 36 types of molecular functions respectively.
Expression levels of arrayed clones gave highly reproducible results, providing confidence in identification of
gene expression patterns. These data demonstrated the power of this new resource to facilitate a greater
understanding into the biological complexities of Trichomonas and coulr be used to develop novel
intervention strategies. Next generation of oligonucleotide arrays will be based on annotated genes of the
Trichomonas genome sequencing project and 70,000 expressed sequenced tags.
CLONING AN EXPRESSION OF A GENE FRAGMENT ENCODING THE FUNCTIONAL
DOMAIN OF CP65 CYSTEINE PROTEINASE INVOLVED IN THE TRICHOMONAS VAGINALIS
CELLULAR DAMAGE.
                           1                                   2                                 1
Eduardo Solano-González, María Elizbeth Alvarez-Sánchez, Victor Hugo Rodríguez-Vargas, Leticia Avila-
                                1                      2*
González, Jaime Ortega-López and Rossana Arroyo
                                                   1                                                 2
Departamento de Biotecnología y Bioingeniería , Departamento de Patología Experimental , Centro de
Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN 2508, Col. San Pedro
Zacatenco, C.P 07360, México D.F., México. E-mail: rarroyo@cinvestav.mx

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs). At least 23 CPs have
been identified by one-and two-dimensional (1-D and 2-D) substrate gel electrophoresis of T. vaginalis
extracts.   A CP of 65 kDa, CP65, with affinity to the surface of HeLa and vaginal epithelial cells was
identified and characterized as being involved in cytotoxicity. CP65 is located on the parasite surface, is
                                                                        o
inhibited by E64, and has an optimal proteolytic activity at pH 5.5 at 37 C and is formed by a single spot with
a pI of 7.2. In an attempt to clone the gene that encode for CP65, a 65 kDa proteinase was purified by
ammonium sulfate fractionation and ion exchange chromatography, tested for proteolytic activity by
substrate gel electrophoresis, for affinity to the surface of HeLa cells by a cell-binding assay, and N-terminal
sequenced. A gene fragment of 618 bp was amplified by PCR using as a sense primer the N-terminal
sequence of the first ten amino acids residues from CP65 and as antisense primer the Asn papain-catalytic
conserved region.    This gene fragment encoded for 206 amino acid residues that corresponded to the
catalytic domain of a CP with 67-76% identity to the reported trichomonad cathepsins-L-like CPs. This gene
fragment was used as a probe in Southern and Northern experiments showing that the tvcp65 gene is a
multicopy gene with a transcript size of 2.3 kb. RT-PCR assays using RNA from parasites grown in high and
low iron concentrations showed that tvcp65 is better transcribed in low iron concentrations. To confirm that
this DNA sequence corresponds to the CP65 proteinase, the 618 bp gene fragment was cloned into the
pET38b(+) expression vector and transformed into BL21/D3 Escherichia coli. The 43 kDa fusion protein was
expressed and purified for antibody production and functional assays.          The recombinant protein was
recognized by the anti-native CP65 antibodies, bound to the surface of HeLa cells, and competed with the
native CP65 for binding. Antibody to the recombinant protein reacted with the trichomonad CP65 proteinase
by Western blot and immunoprecipitation assays, and inhibited trichomonal cytotoxicity in a concentration-
dependent manner. This data strongly suggest that the functional and cell-binding domains of CP65 are
localized in the 206 amino acid residues encoded by the 618 bp tvcp65 gene fragment.
CHARACTERIZATION OF THE 5S RIBOSOMAL RNA GENE IN Trichomonas vaginalis:
ANALYSIS PUTATIVE RNA POLYMERASE III PROMOTER ELEMENTS.
                            1                      1                    2                            1
Ana Lilia Torres-Machorro , Roberto Hernández , Joaquín Sánchez , Imelda López-Villaseñor
1
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad
                                                                                                         2
Nacional Autónoma de México. Apartado Postal 70-228, C.P. 04510, México D.F., México. Facultad de
Medicina, Universidad Autónoma del Estado de Morelos. Avenida Universidad 1001, Cuernavaca Morelos,
C.P. 62210, México.

Trichomonas vaginalis is a parasitic anaerobic protozoa, etiologic agent of human trichomonosis.
Phylogenetic analyses based on rRNA and actin sequences place this organism among the earlier
eukaryotic branches. Consistent with its early evolution, some trichomonad ribosomal features are
reminiscent of prokaryotes, for example, its ribosomes have a sedimentation coefficient of 70S , more
related to their eubacterial counterparts than to the eukaryotic ones.

The typical eukaryotic ribosome is composed of the ribosomal RNAs (rRNA) 18S, 5.8S, 28S and 5S. The
first three molecules are generally all encoded in a ribosomal main transcription unit (rDNA) and are
transcribed by RNA polymerase I. In contrast, the 5S rRNA gene is transcribed by RNA polymerase III and is
not usually linked to the rDNA unit. The 5S rRNA is found in all eukaryotic ribosomes, having important roles
in ribosome biogenesis, peptidil-transferase activity, translational fidelity and large subunit stability.

In this work we characterized the T. vaginalis 5S rRNA gene and determined its relatedness, in nucleotide
sequence and in potential folding, to eukaryotic and bacterial 5S rRNAs. The putative coding region is 118
bases long and contains the typical RNA polymerase III promoter elements (internal to the coding region)
such as Box A, intermediate element and Box C sequences (ICR). In addition, we identified other regulatory
element such as a putative TATA-box and termination sequences. The previous characterization is of
particular interest because although RNA polymerase I and RNA polymerase II promoters for T. vaginalis
have been documented, no RNA polymerase III promoter has yet been identified in this early diverging
organism.

The ribosomal genes are reiterated In the eukaryotic genomes. The copy number rate of the main
transcription unit vs 5S rRNA genes of different organisms is extremely variable, ranging from 0.0625 in
Trypanosoma cruzi to 13.3 in Euglena gracilis. We estimated the rate of T. vaginalis ribosomal genes at
                                                                                                7
0.04. With these analyses, we also estimated the T. vaginalis genomic size in 2.5 x 10 bp, which agrees
with Wang’s (1985) original genomic size proposal.
STRUCTURAL            CHARACTERIZATION             AND     LOCALIZATION        OF    ANNEXIN        E1    IN
TROPHOZOITES OF GIARDIA LAMBLIA

Anke Vahrmann, Dennis Priess, Anna Szkodowska, Tilly Bakker-Grunwald, and Henning Scholze

Department of Biology/Chemistry, Biochemistry, University of Osnabrueck, Germany

Annexins represent a multigene family of eukaryotic proteins with various, but in detail mostly unknown
functions. The genome of the parasitic protozoon Giardia lamblia contains at least 21 genes encoding the
annexin-homologous alpha-giardins. From these, we have identified annexin E1 (?14-giardin) in a cell
extract of trophozoites of G. lamblia, which, in contrast to its homologous ?1- and ?2-giardins (annexinE2
and E3), is equipped with a true type II Ca2+-binding motif (GXGTD{38}D) in its second domain and a further
rudimentary one in the fourth annexin repeat. Furthermore, in the C-terminal region of annnexin E1, some
elements typically occurring in ?-giardins are conspicuous, such as an ITG/AM- and a KXXYK-motif, and a
tryptophan residue followed by a charged residue next to the C-terminus. These sequence elements, whose
physiological significance is still unknown, are not conserved in annexins of higher eukaryotes. According to
molecular modelling of annexin E1 based on known annexin structures, these residues are positioned next
to the concave side of the molecule, which in other annexins is orientated to the cytoplasmic face of the
molecule. Western blot analyses of isolated giardial flagella revealed a strong cross-reaction with anti-
annexin E1 antibodies. This finding was confirmed by immunocytochemical analyses, which show that
annexin E1 is a constitutive part of the eight giardial flagella as well as of the median body of the
trophozoites, but does not directly interact with cytoskeletons.

With the aid of affinity chromatography of a trophozoite extract on immobilized annexin E1, we identified
several proteins as potential interaction partners of annexin E1 by mass spectroscopy. Besides the ?2- and
?7.3-giardins and annexin E1 itself - electron microscopy of the purified recombinant annexin E1 suggests
that the protein is able to self assemble into fibrils- there was a protein with two domains, an N-terminal
Ser/Thr-kinase and a C-terminal part consisting of several ankyrin-repeats. Whereas Ser/Thr-kinases
regulate their target proteins by ATP-dependent phosphorylation, the ankyrin repeat is one of the most
abundant protein-protein interaction motifs. We hypothesize that annexin E1 may function as a Ca2+-
regulated structural element linking the phospholipids bilayer to the underlying axoneme and possibly being
involved in cell motility.
IDENTIFICATION OF TRICHOMONADS IN HUMAN TISSUES BY MOLECULAR TOOLS
                             1                    2                   3                    3
Christophe DUBOUCHER , Stéphanie CABY , Nausicaa GANTOIS , Magalie CHABE , Isabelle DURAND-
      3                          2                             4                       2                     3
JOLY , Christophe NOËL , Pilar-DELGADO-VISCOGLIOSI , Monique CAPRON , Eduardo DEI-CAS ,
                 5                           2
Gerard MOREL , and Eric VISCOGLIOSI
1                                                                         2
Laboratory of Pathology, Saint-Germain-en-Laye Hospital, France; Inserm U547, Institut Pasteur of Lille,
          3                                                                        4
France; Laboratory of Ecology and Parasitism, Institut Pasteur of Lille, France; Department of Water and
                                                  5
Environment, Institut Pasteur of Lille, France; CNRS UMR 5123, University Claude Bernard, Lyon, France.

Since the discovery of Trichomonas vaginalis in 1836, trichomonads have little been involved in human
pathology. Excluding studies dealing with genital trichomoniasis, only single cases of trichomoniasis outside
the genitourinary tract were reported. It mainly involved the respiratory tract and were often linked to
Trichomonas tenax, a species considered as a harmless saprophyte of the mouth in humans. To our
knowledge, T. vaginalis had not been reported in the lungs, apart from exceptional cases in enfants after
contamination during delivery.
Recently, we have identified T. vaginalis using molecular tools, together with Pneumocystis parasites, in the
bronchoalveolar lavage fluid (BALF) of an adult patient with AIDS (1). Interestingly, the closely related
species, Tritrichomonas foetus from bovids, has also been identified in the lungs of an immunocompromised
patient again in association with Pneumocystis organisms (Duboucher et al., in preparation).
To investigate the incidence of trichomonads and Pneumocystis in lungs, sixty-six BALs from
immunocompromised patients with pneumocystosis were retrospectively examined microscopically (2).
Strikingly, trichomonads were found as coinfecting agents in 60% of BALF samples. The apparent
correlation between the abundance of Pneumocystis and trichomonad populations strongly suggested that a
local environment containing Pneumocystis seems to be necessary to the development of these anaerobic
protozoans in addition to, or instead of, immunodepression.
Physicians are well aware of the presence of trichomonads in the genitourinary tract, intestine and oral cavity
but not outside their natural habitat, such as lungs. During their adhesion to host cells, these parasites
undergo deep cytoskeletal modifications that involve a change from a flagellate to an ameboid form.
Numerous parasitic cells with a large nucleus mimicking human cells, as well as other unusual features,
such as cells with 2 or more nuclei, are observable in some BALF samples. Such pleomorphism renders
trichomonad identification on cytologic slides difficult as long as the conception of trichomonad morphology
is restricted to that of the flagellated stage.
We are currently developing chromogenic in situ hybridization (CISH) techniques in order to identify
trichomonads in unusual locations. Small subunit rRNA gene sequences are already known for the
trichomonad species found or that may be found in human. SSU rRNA probes have been designed allowing
the detection of trichomonads at the species level and preliminary data concerning this approach are
presented. These tools will allow us to define the real incidence of trichomonads in unusual locations,
invasion pathway, and pathogenicity (observation of alveolar damage for instance).
(1) Duboucher C et al. Hum Pathol. 2003; 34: 508-11.
(2) Duboucher C et al. Acta Cytol. 2005; 49: 273-7.

				
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Description: Aerobic exercise anaerobic exercise is relatively speaking. During exercise, the body's metabolism is accelerated to speed up the metabolic needs more energy. The body's energy through the body of sugar, protein and fat catabolism come. When not in the exercise, such as jogging, playing badminton, dancing, etc., the body's supply of energy mainly from aerobic metabolism of fat. To fat as the main supply of energy aerobic exercise aerobic exercise is what we say. When we engage in very intense exercise, or the rapid outbreak, such as weightlifting, 100 m sprint, wrestling, etc., then the body needs a lot of energy in an instant, and in normal circumstances, aerobic metabolism can not meet the body at this time demand, so the conduct of anaerobic metabolism of sugar, to rapidly produce large amounts of energy. This state is anaerobic exercise.