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Molecular and cellular characterisation of Burkholderia

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Molecular and cellular characterisation of Burkholderia Powered By Docstoc
					Molecular and cellular characterisation

        of Burkholderia pseudomallei

          interactions with host cells




A thesis submitted in fulfillment of the requirements of the degree of

      Doctor of Philosophy in the School of Medical Science,

            Griffith University, Gold Coast, Queensland.



                          December, 2005




  By Justin Andrew Boddey, B.Biomed.Sc.(Hons.)
Synopsis


      Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of

serious and often-fatal diseases, collectively termed melioidosis. B. pseudomallei is

capable of interacting specifically with host cells; it can adhere, invade, and survive and

replicate intracellularly and induce the formation of multinucleated giant cells (MNGCs).

While recent work has begun characterising these interactions at the molecular level, much

is still unknown. In this work, B. pseudomallei interactions with eukaryotic cells were

investigated at the cellular and molecular level. Many putative loci were studied by, firstly,

generating isogenic mutant strains lacking a gene or locus of interest; and secondly, by

characterising each mutant strain using a number of in vitro and in vivo models. These

included models for adherence to eukaryotic cells, microcolony development, biofilm

formation, twitching motility, bacterial cell aggregation, invasion of eukaryotic cells,

actin-based motility, intracellular proliferation, multinucleated giant cell (MNGC)

development and characterisation, gene expression, and virulence in vivo.

      Three putative type IV pilus (TFP) systems were identified in nine loci in the

B. pseudomallei K96243 genome; type IVA, IVB, and IVB Flp pilus systems. Studies

revealed that TFP are produced on the surface of B. pseudomallei K96243 but that TFP

play only a minor role in adherence to eukaryotic cells using the assays described.

However, collaborators showed that B. pseudomallei K96243 uses the type IVA pilin,

PilA, to adhere to eukaryotic cells using a different adherence assay. Interestingly, two

strains of B. pseudomallei adhered to eukaryotic cells to different degrees and used TFP

(pilA) differently. B. pseudomallei K96243 adhered significantly less to four cell lines than


                                                                                              ii
B. pseudomallei 08. Furthermore, adherent microcolony formation was shown to be a

temperature-dependent phenotype that enhanced bacterial association with eukaryotic

cells; however, while B. pseudomallei 08 required pilA to form microcolonies, strain

K96243 did not form microcolonies, and cell association was markedly reduced for

K96243 relative to strain 08.

      TFP were not required for the formation of biofilms on PVC; however, type IVA and

IVB Flp pili were required for optimal virulence in BALB/c mice using the intranasal route

of infection, indicating the importance of TFP for B. pseudomallei survival in vivo.

      This work also identified that growth temperature, growth medium, and association

with eukaryotic cells were important regulatory signals for adherence/cell association,

microcolony formation, biofilm development, and TFP (pilA) expression.

      A homologue of eukaryotic “senescence marker protein-30” (SMP-30) was

identified in B. pseudomallei 08 and K96243 and was termed Lfp1 (for lactonase family

protein 1). lfp-1 is located within a genomic island and is conserved in both prokaryotes

and eukaryotes. A homologue of lfp-1 (lfp-2) was also present in B. pseudomallei K96243.

Though sharing considerable homology, prokaryotic and eukaryotic homologues of lfp-1

were shown to be phylogenetically distinct. Expression of lfp-1 mRNA by B. pseudomallei

08 was significantly increased in association with eukaryotic cells, relative to maintenance

media alone; however, lfp-1 was not required for adherence, invasion, intracellular

proliferation, actin-based motility, or cell fusion by B. pseudomallei. Importantly though,

B. pseudomallei 08-infected macrophage-like cells rapidly fused into MNGCs, assuming

an lfp-1-specific osteoclast-like pattern of gene expression that was distinct from

B. thailandensis-infected MNGCs, and the process may be different to the LPS-dependent


                                                                                            iii
mechanisms of osteoclast-like cell induction described previously for other bacteria.

B. pseudomallei-induced MNGC formation correlated with potent increases in mRNA

levels for the cell fusion and multinucleation factors MCP-1, CCL9/MIP-1γ, RANTES,

and NFATc1 in B. pseudomallei-infected cells, implicating these molecules in cell fusion.

B. pseudomallei-induced osteoclast-like MNGCs could not authentically resorb dentine;

however, regions of apparently demineralised dentine were observed, suggesting a defect

in the excavation of the organic phase of bone, analogous to that observed in CTSK

knockout mice. Finally, an lfp-1 null mutant was significantly attenuated for virulence in

both the Syrian hamster model and the BALB/c inhalation model, indicating a role for

lfp-1 in virulence.




                                                                                         iv
Table of Contents



   Synopsis ............................................................................................................................ ii

   Acknowledgements ........................................................................................................ xiv

   Statement of Originality.................................................................................................. xv

   List of Figures ................................................................................................................ xvi

   List of Tables ................................................................................................................. xix

   List of Abbreviations ...................................................................................................... xx



Chapter One: Introduction......................................................................................... 1

   1.1 The disease: melioidosis ............................................................................................. 2

      1.1.1 Epidemiology ....................................................................................................... 2

          1.1.1.1 Endemicity and incidence of melioidosis ..................................................... 2

          1.1.1.2 Geographical distribution of melioidosis ...................................................... 4

          1.1.1.3 Modes of infection ........................................................................................ 4

      1.1.2 Clinical features of melioidosis............................................................................ 6

      1.1.3 Diagnosis of melioidosis ...................................................................................... 8

      1.1.4 Treatment of melioidosis ..................................................................................... 9

      1.1.5 Predispositions to melioidosis............................................................................ 11

      1.1.6 Melioidosis in mammals .................................................................................... 12

      1.1.7 Animal models of melioidosis ........................................................................... 12

      1.1.8 Melioidosis: under-recognised or an emerging global disease? ........................ 17

      1.1.9 Melioidosis and the December 26, 2004, Tsunamis .......................................... 18

                                                                                                                                       v
  1.2 The aetiological agent: Burkholderia pseudomallei ................................................. 19

     1.2.1 General bacteriology .......................................................................................... 19

     1.2.2 Environmental microbiology ............................................................................. 20

     1.2.3 Molecular and cellular mechanism of B. pseudomallei virulence ..................... 21

         1.2.3.1 Toxins.......................................................................................................... 21

         1.2.3.2 Other secreted proteins................................................................................ 22

         1.2.3.3 Type III secretion systems (TTSSs)............................................................ 23

         1.2.3.4 Surface polysaccharides .............................................................................. 24

         1.2.3.5 Flagella and motility ................................................................................... 25

         1.2.3.6 Quorum sensing .......................................................................................... 25

         1.2.3.7 Biofilms....................................................................................................... 26

         1.2.3.8 Adherence to cultured cells......................................................................... 27

         1.2.3.9 Invasion of cultured cells ............................................................................ 28

         1.2.3.10 Survival and proliferation within cultured cells........................................ 29

         1.2.3.11 Actin-based motility.................................................................................. 30

         1.2.3.12 Cell fusion and multinucleated giant cell (MNGC) formation ................. 30

     1.2.4 B. pseudomallei as a potential biological weapon ............................................. 31

  1.3 Research objectives................................................................................................... 32



Chapter Two: Materials and Methods ................................................................. 33

  2.1 Materials.................................................................................................................... 34

     2.1.1 Chemicals........................................................................................................... 34

     2.1.2 Enzymes ............................................................................................................. 35

                                                                                                                                 vi
   2.1.3 Bacterial culture medium ................................................................................... 36

   2.1.4 Cell culture medium and supplements ............................................................... 36

   2.1.5 Bacterial strains and plasmids............................................................................ 37

   2.1.6 Oligonucleotides ................................................................................................ 41

   2.1.7 Cell Lines ........................................................................................................... 43

2.2 Methods..................................................................................................................... 44

   2.2.1 in silico analyses ................................................................................................ 44

   2.2.2 Bacterial growth conditions and storage ............................................................ 45

   2.2.3 Preparation of genomic DNA............................................................................. 45

   2.2.4 Preparation of plasmid DNA.............................................................................. 46

   2.2.5 Preparation of total RNA ................................................................................... 47

   2.2.6 Preparation of first-strand cDNA ....................................................................... 47

   2.2.7 Agarose gel electrophoresis ............................................................................... 48

   2.2.8 Spectrophotometric quantification of DNA and RNA concentration ................ 48

   2.2.9 DNA purification from agarose gels .................................................................. 49

   2.2.10 Automated DNA sequencing ........................................................................... 49

   2.2.11 Restriction endonuclease digestion of DNA .................................................... 50

   2.2.12 Ligation of DNA .............................................................................................. 50

   2.2.13 Preparation of chemically competent cells ...................................................... 50

   2.2.14 Preparation of electro-competent cells............................................................. 51

   2.2.15 Transformation of E. coli by heat shock .......................................................... 51

   2.2.16 Transformation of E. coli by electroporation................................................... 51

   2.2.17 Rapid plasmid screening of transformants....................................................... 52


                                                                                                                             vii
2.2.18 Conjugation...................................................................................................... 52

2.2.19 Polymerase chain reaction (PCR) .................................................................... 53

2.2.20 Southern blot and DNA denaturation and fixation .......................................... 53

2.2.21 Synthesis, hybridisation, and detection of radio-labelled nucleotide probes ... 54

2.2.22 Estimation of DNA molecular weight following Southern blot ...................... 55

2.2.23 Mutagenesis in B. pseudomallei....................................................................... 55

2.2.24 Maintenance of cultured cell lines ................................................................... 57

2.2.25 Storage and resuscitation of cell lines.............................................................. 58

2.2.26 Enumeration of cultured cells per growth area ................................................ 58

2.2.27 Fixation of samples .......................................................................................... 59

2.2.28 Giemsa staining of samples.............................................................................. 59

2.2.29 TRAP staining of samples................................................................................ 59

2.2.30 Immunolabelling of samples ............................................................................ 60

2.2.31 Negative staining of samples ........................................................................... 60

2.2.32 Sputter coating of samples ............................................................................... 61

2.2.33 Mounting of samples........................................................................................ 61

2.2.34 Light, confocal, and electron microscopy ........................................................ 61

2.2.35 Bacterial biofilm assay..................................................................................... 62

2.2.36 Bacterial cell aggregation assay ....................................................................... 62

2.2.37 Twitching motility assays ................................................................................ 63

2.2.38 Bacterial adherence assays............................................................................... 63

2.2.39 Bacterial microcolony assays........................................................................... 65

2.2.40 Bacterial invasion assays.................................................................................. 66


                                                                                                                    viii
     2.2.41 Bacterial intracellular proliferation, MNGC formation, and actin-based

     motility assays............................................................................................................. 66

     2.2.42 Bacterial growth assays and cell-line infection assays for quantitative

     molecular analysis....................................................................................................... 67

     2.2.43 Assay for resorption of dentine ........................................................................ 68

     2.2.44 Quantitative real-time PCR (Q-PCR) .............................................................. 68

     2.2.45 RNA/cDNA preparation, microarray hybridisation, and analyses................... 69

     2.2.46 Animal infection studies .................................................................................. 70

     2.2.47 Statistical analyses ........................................................................................... 71



Chapter Three: Adherence, microcolony formation, and biofilm

development by Burkholderia pseudomallei........................................................ 73

  3.1 Introduction............................................................................................................... 74

  3.2 Results and discussion .............................................................................................. 75

     3.2.1 B. pseudomallei K96243 adherence: a comparative analysis with strain 08 ..... 75

     3.2.2 B. pseudomallei adherence is enhanced by centrifugation................................. 77

     3.2.3 Microscopic assessment of B. pseudomallei adherence/association: role

     of adherence, microcolonies, and temperature............................................................ 78

     3.2.4 B. pseudomallei forms biofilms: a temperature- and media-dependent

     phenotype .................................................................................................................... 84

     3.2.5 Temperature and culture media are important regulatory signals for

     B. pseudomallei ........................................................................................................... 88

  3.3 Conclusions ............................................................................................................... 90

                                                                                                                                 ix
  3.4 Acknowledgements ................................................................................................... 91



Chapter Four: Identification of nine type IV pilus loci comprising

three type IV pilus systems in Burkholderia pseudomallei ............................ 92

  4.1 Introduction............................................................................................................... 93

  4.2 Results and discussion .............................................................................................. 95

     4.2.1 B. pseudomallei K96243 harbors nine type IV pilus (TFP) loci........................ 95

     4.2.2 B. pseudomallei K96243 TFP loci contain twelve subunit genes...................... 97

     4.2.3 B. pseudomallei K96243 TFP loci comprise three TFP systems ..................... 102

         4.2.3.1 The P. aeruginosa-like type IVA pil system in B. pseudomallei.............. 103

         4.2.3.2 The R64-like type IVB pil system in B. pseudomallei.............................. 108

         4.2.3.3 The A. actinomycetemcomitans/C. crescentus-like type IVB flp/cpa

         pilus system in B. pseudomallei ............................................................................ 115

     4.2.4 Presence and organisation of TFP loci in B. mallei and B. thailandensis........ 123

  4.3 Conclusions ............................................................................................................. 128

  4.4 Acknowledgements ................................................................................................. 128



Chapter Five: Mutagenesis and functional characterisation of

three TFP systems in Burkholderia pseudomallei ........................................... 129

  5.1 Introduction............................................................................................................. 130

  5.2 Results and discussion ............................................................................................ 131

     5.2.1 Unmarked deletion of B. pseudomallei TFP loci ............................................. 131

     5.2.2 Transmission electron microscopy (TEM) studies on B. pseudomallei........... 139
                                                                                                                                x
      5.2.3 Role of B. pseudomallei TFP loci in adherence to human cells and

      adherent microcolony formation ............................................................................... 142

          5.2.3.1: Characterising TFP systems in B. pseudomallei K96243 ........................ 142

          5.2.3.2: Characterising pilA in B. pseudomallei 08............................................... 145

      5.2.4 Microcolony formation by B. pseudomallei 08 in the absence of cultured

      cells requires pilA...................................................................................................... 149

      5.2.5 B. pseudomallei 08 pilA mRNA expression is modulated by temperature,

      media, and association with eukaryotic cells ............................................................ 152

          5.2.5.1 Effects of temperature and agar ................................................................ 152

          5.2.5.2 Effects of temperature, tissue culture media, and eukaryotic cells ........... 153

      5.2.6 Role of B. pseudomallei TFP loci in biofilm formation................................... 155

      5.2.7 Role of B. pseudomallei TFP loci in cell aggregation ..................................... 158

      5.2.8 Role of B. pseudomallei TFP loci in twitching motility .................................. 159

      5.2.9 Role of B. pseudomallei TFP loci in virulence in vivo .................................... 161

          5.2.9.1 Virulence in nematode worms .................................................................. 161

          5.2.9.2 Virulence in BALB/c mice........................................................................ 163

      5.2.10 Micro-array analysis of B. pseudomallei TFP expression ............................. 167

   5.3 Conclusions ............................................................................................................. 170

   5.4 Acknowledgements ................................................................................................. 172



Chapter Six: Identification and characterisation of lfp-1 in

Burkholderia pseudomallei which encodes a homologue of the

eukaryotic protein SMP-30..................................................................................... 173
                                                                                                                               xi
6.1 Introduction............................................................................................................. 174

6.2 Results and discussion ............................................................................................ 175

   6.2.1 Identification of lfp-1 in B. pseudomallei ........................................................ 175

   6.2.2 Sequence analysis of B. pseudomallei lfp-1 and Lfp1 ..................................... 177

   6.2.3 B. pseudomallei K96243 contains a second SMP-30 homologue ................... 178

   6.2.4 Distribution of lfp-1 and Lfp1 in B. pseudomallei strains and

   Burkholderia species................................................................................................. 180

   6.2.5 Lfp1 homologues are wide-spread in prokaryotes and are highly

   conserved amongst prokaryotes and eukaryotes ....................................................... 181

   6.2.6 Unmarked and in-frame deletion of lfp-1 in B. pseudomallei 08..................... 186

   6.2.7 Complementation of B. pseudomallei lfp-1 ..................................................... 187

   6.2.8 Examining the role of lfp-1 in B. pseudomallei growth in

   calcium-limiting media ............................................................................................. 189

   6.2.9 lfp-1 is expressed in association with macrophage-like cells........................... 190

   6.2.10 Examining the role of lfp-1 in B. pseudomallei interactions with

   host cells.................................................................................................................... 191

   6.2.11 Examining the role of lfp-1 in virulence in vivo ............................................ 194

       6.2.11.1 Insertional inactivation of lfp-1............................................................... 194

       6.2.11.2 Virulence in Syrian hamsters .................................................................. 195

       6.2.11.3 Virulence in BALB/c mice ..................................................................... 196

6.3 Conclusions ............................................................................................................. 197

6.4 Acknowledgements ................................................................................................. 198




                                                                                                                              xii
Chapter Seven: Characterisation of Burkholderia

pseudomallei-infected macrophage-like cells.................................................... 199

   7.1 Introduction............................................................................................................. 200

   7.2 Results and discussion ............................................................................................ 201

       7.2.1 MNGC formation in RAW264.7 cells infected with B. pseudomallei

       08 and JAB136 and B. thailandensis E264............................................................... 201

       7.2.2 B. pseudomallei 08-infected RAW264.7 cells rapidly produce

       tartrate-resistant acid phosphatase (TRAP)............................................................... 205

       7.2.3 B. pseudomallei–infected RAW264.7 cells rapidly express osteoclast

       markers and chemokines, and NFATc1: a process involving lfp-1 .......................... 209

       7.2.4 B. pseudomallei-infected RAW264.7 cells are not authentic osteoclasts ........ 216

   7.3 Conclusions ............................................................................................................. 222

   7.4 Acknowledgements ................................................................................................. 223



Chapter Eight: Summary and Conclusions...................................................... 224

   8.1 Summary and conclusions ...................................................................................... 225



Chapter Nine: References ....................................................................................... 235



Appendix I: Research papers published and prepared during

candidature................................................................................................................... 273



                                                                                                                             xiii
Acknowledgements


      I am very grateful for an Australian Postgraduate Award. I acknowledge the essential

collaborations that made this work possible: I thank Dr. Angela Essex-Lopresti, and her

colleagues at Defence Science Technology Laboratory (DSTL), for collaborating on type

IV pili (TFP) studies, and Dr. Ricky Ulrich and his colleagues at both the United States

Army Medical Research Institute of Infectious Diseases (USAMRIID), and the Institute

for Genomic Research (TIGR), for collaborating on both TFP and Lfp1 characterisation. I

also am very grateful to Mr. Chris Day, Mrs. Carie-Anne Logue, and Mr. Cameron Flegg,

for their essential collaborative roles whilst investigating TFP and Lfp1. Collaborator’s

contributions are specifically outlined in the Acknowledgements section at the end of

relevant chapters. I also thank Dr. Julian Parkhill at the Sanger Institute and Dr. William

Nierman at TIGR for providing access to genome sequences prior to publication.

      I extend a sincere thank you to my supervisors, Professor Ifor Beacham and Dr. Ian

Peak, for their guidance, support, inspiration, and friendship throughout my time as a Ph.D

student. I also thank Dr. Nigel Morrison for his support. The technical assistance and

valued friendship of members of the Beacham/Peak laboratory, namely, Nat Brown,

Carie-Anne Logue, Connor McCarthy, Liz Allwood, Isabelle Faglin, Cameron Flegg, and

Yveth Cossart, and the members of the Science 2, 3.22 laboratory, is greatly appreciated.

      I thank Claire, Emma, Rick, Kai, Mia Mia, and Lachlan for their excitement, endless

love, and support. Finally, I sincerely thank my wonderful girlfriend Catherine, for her

love, support, understanding, inspiring mind, and for taking intermission from her

doctorate and moving interstate so that this work could happen.


                                                                                           xiv
Statement of Originality


      This work has not previously been submitted for a degree or diploma in any

university. To the best of my knowledge and belief, this thesis contains no material

previously published or written by another person except where due reference is made in

the thesis itself. Material arising from the direct collaboration of myself with other

scientists has been included in this thesis with the full knowledge and consent of

collaborators. Collaborator’s contributions are indicated in the Acknowledgements sections

of this thesis.




Justin A. Boddey

30.11.2005




                                                                                         xv
List of Figures


Figure 3.1: Adherence of B. pseudomallei to different cell lines........................................ 76

Figure 3.2: Centrifugation enhances B. pseudomallei adherence ....................................... 78

Figure 3.3: Microscopy studies of B. pseudomallei adherence/cell-association................. 82

Figure 3.4: Biofilm formation by B. pseudomallei ............................................................. 86

Figure 3.5: Static growth of B. pseudomallei K96243........................................................ 88

Figure 4.1: ClustalW alignments of the N-terminal amino acids of representative

      B. pseudomallei K96243 pilins to other type IVA and IVB pilins ............................. 97

Figure 4.2: Predicted leader peptide of PilA from B. pseudomallei K96243 ................... 100

Figure 4.3: Genetic organization of the type IVA-associated pil loci in

      P. aeruginosa PAO1 (Pa) and B. pseudomallei K96243 (Bp).................................. 105

Figure 4.4: Genetic organization of the type IVB-associated pil loci from E. coli

      plasmid R64 (R64) and B. pseudomallei K96243 (Bp) ............................................ 110

Figure 4.5: Genetic organization of the flp/cpa loci in A. actinomycetemcomitans

      (Aa), C. Crescentus (Cc), and B. pseudomallei (Bp) ................................................ 117

Figure 5.1: B. pseudomallei K96243 TFP loci targeted for individual deletion

      mutagenesis ............................................................................................................... 132

Figure 5.2: Schematic overview of unmarked deletion mutagenesis in

      B. pseudomallei ......................................................................................................... 135

Figure 5.3: Southern blot analyses of B. pseudomallei strains acquired during

      mutagenesis of TFP loci............................................................................................ 137




                                                                                                                              xvi
Figure 5.4: Southern blot analyses of B. pseudomallei K96243 strains acquired

     during double deletion mutagenesis of TFP loci ...................................................... 139

Figure 5.5: Electron microscopy of B. pseudomallei K96243 producing pili .................. 141

Figure 5.6: Role of TFP in B. pseudomallei K96243 adherence/cell association............. 144

Figure 5.7: Role of pilA in B. pseudomallei 08 adherence/cell association...................... 148

Figure 5.8: Role of pilA in B. pseudomallei 08 microcolony formation in the

     absence of human cells.............................................................................................. 151

Figure 5.9: Expression of pilA mRNA in B. pseudomallei 08 .......................................... 155

Figure 5.10: Role of TFP in B. pseudomallei biofilm formation ...................................... 157

Figure 5.11: Assessment of B. pseudomallei K96243 twitching motility......................... 160

Figure 5.12: Role of TFP2 in B. pseudomallei K96243 virulence in C. elegans.............. 162

Figure 5.13: Role of TFP2 in B. pseudomallei K96243 virulence in BALB/c mice ........ 164

Figure 5.14: Role of TFP loci in B. pseudomallei virulence in BALB/c mice ................. 167

Figure 6.1: B. pseudomallei Lfp1 may be a lipoprotein.................................................... 178

Figure 6.2: B. pseudomallei K96243 Lfp1 and Lfp2 are divergent .................................. 179

Figure 6.3: Southern blot analysis of lfp-1 distribution in B. pseudomallei strains

     and other Burkholderia species................................................................................. 181

Figure 6.4: Prokaryotic and eukaryotic homologues of Lfp1 are highly conserved......... 184

Figure 6.5: Phylogenetic relationships between prokaryotic and eukaryotic Lfp1

     homologues ............................................................................................................... 185

Figure 6.6: Southern blot analyses of B. pseudomallei strains acquired during

     mutagenesis of lfp-1 .................................................................................................. 187




                                                                                                                           xvii
Figure 6.7: Assessment of lfp-1 expression in the lfp-1 complemented strain

     JAB136C ................................................................................................................... 188

Figure 6.8: Growth of B. pseudomallei strains in calcium-limiting media....................... 189

Figure 6.9: lfp-1 is expressed in association with RAW264.7 cells.................................. 190

Figure 6.10: Adherence, invasion, intracellular proliferation, and actin-based

     motility of B. pseudomallei 08 and JAB136 ............................................................. 193

Figure 6.11: lfp-1 is necessary for optimal virulence in Syrian hamsters......................... 196

Figure 6.12: lfp-1 is necessary for optimal virulence in BALB/c mice ............................ 197

Figure 7.1: MNGC formation by B. pseudomallei 08 and JAB136 and

     B. thailandensis E264 ............................................................................................... 203

Figure 7.2: TRAP production in B. pseudomallei-infected RAW264.7 cells................... 207

Figure 7.3: Expression of osteoclast markers, chemokines, and NFATc1 by

     infected RAW264.7 cells .......................................................................................... 211

Figure 7.4: Effects of B. pseudomallei 08-infected RAW264.7 cells on dentine ............. 218




                                                                                                                           xviii
List of Tables


Table 2.1: Bacterial strains and plasmids used in this study............................................... 37

Table 2.2: Oligonucleotides used in this study ................................................................... 41

Table 2.3: Cell lines used in this study ............................................................................... 43

Table 4.1: Summary of predicted TFP loci identified in the B. pseudomallei

      K96243 genome .......................................................................................................... 96

Table 4.2: Summary of TFP subunits identified in the B. pseudomallei K96243

      genome ........................................................................................................................ 99

Table 4.3: Genetic organization of B. pseudomallei TFP loci present in B. mallei

      ATCC23344 and B. thailandensis E264 ................................................................... 125

Table 5.1: Summary of TFP loci mutations generated in B. pseudomallei....................... 131

Table 5.2: Summary of investigations into pili production by B. pseudomallei............... 141

Table 5.3: Micro-array analyses of B. pseudomallei 1026b TFP loci expression

      at 25 ºC and 37 ºC at logarithmic- and stationary-phase of growth in LB

      medium...................................................................................................................... 169

Table 6.1: Ten most similar prokaryotic and eukaryotic homologues of

      B. pseudomallei Lfp1 ................................................................................................ 182




                                                                                                                                 xix
List of Abbreviations



AEMF                    Australian Electron Microscopy Facility

Ap                      ampicillin

Asialo GM1              gangliotetraosylceramide

Asialo GM2              gangliotriaosylceramide

ATCC                    American Type Culture Collection

ATP                     adenosine triphosphate

BSA                     bovine serum albumin

bp                      base pairs

C-                      carboxy-

CDC                     Centers for Disease Control and Prevention

cDNA                    copy deoxyribonucleic acid

CFU                     colony forming units

CCL9/MIP-1γ             macrophage inflammatory protein 1 gamma

CLT                     cytolethal toxin

Cm                      chloramphenicol

CTAB                    cetyltrimethylammonium bromide

CTR                     calcitonin receptor

CTSK                    cathepsin K

dCTP                    deoxycytosine triphosphate

dNTPs                   deoxyribonucleotides

Da                      Daltons

                                                                     xx
DC-STAMP   dendritic cell-specific transmembrane protein

DEPC       diethylpyrocarbonate

DMSO       dimethylsulphoxide

DNA        deoxyribonucleic acid

DSTL       Defence Science Technology Laboratory

EDTA       ethylenediaminetetraacetic acid

EGTA       ethyleneglycol-bis-(β-aminoethylether)N,N’-

           tetraacetic acid

EPEC       enteropathogenic Escherichia coli

EPS        exopolysaccharide

FBGC       foreign-body giant cell

FCS        foetal calf serum

G-CSF      granulocyte colony stimulating factor

GI         genomic island

GLC-FAME   gas-liquid chromatography analysis of fatty acid

           methyl esthers

Gm         gentamicin

GM-CSF     granulocyte macrophage colony stimulating factor

HIV        human immuno-deficiency virus

IgA        Immunoglobulin A

IgG        Immunoglobulin G

IgM        Immunoglobulin M

IHA        indirect haemagglutination

                                                              xxi
IPM      imipenem

Kbp      kilo base pairs

Km       kanamycin

LAP      Listeria adhesion protein

LB       Luria-Bertani (medium)

LPS      lipopolysaccharide

Mbp      mega base pairs

MCP-1    monocyte chemotactic protein 1

M-CSF    macrophage colony stimulating factor

MEM      minimum essential medium

MFR      macrophage fusion receptor

MNGC     multinucleated giant cell

MOI      multiplicity of infection

MOPS     3-[N-morpholinopropane]-sulfonic acid

MQ       MilliQ (water)

mRNA     messenger ribonucleic acid

N-       amino-

NFATc1   nuclear factor of activated T-cells cytoplasmic 1

NIAID    United States National Institute of Allergy and

         Infectious Diseases

NT       nick translation

N-WASP   Neural Wiskott-Aldrich syndrome protein

nt       nucleotides


                                                             xxii
ntp      nucleotide position

OD       optical density

OPS      O-antigenic polysaccharide

ORF      open reading frame

PBS      phosphate-buffered saline

PCR      polymerase chain reaction

PLC      phospholipase C

PVC      polyvinyl chloride

Q-PCR    quantitative real-time polymerase chain reaction

RANKL    receptor activator of nuclear factor-κβ ligand

rRANKL   recombinant receptor activator of nuclear factor-

         κβ ligand

RANTES   regulated on activation normal T cell expresses

         and secreted

RNA      ribonucleic acid

rRNA     ribosomal ribonucleic acid

RT       reverse transcriptase

rpm      revolutions per minute

SDS      sodium dodecylsulphate

SEM      standard error of the mean

Sm       streptomycin

SMP-30   senescence marker protein-30

SNARE    soluble NSF-attachment protein receptor

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SSC        sodium chloride/sodium citrate

STEC       Shiga-toxigenic Escherichia coli

TAE        Tris/acetate/ethylene diamine tetraaceticacid

TBE        Tris/borate/ethylene diamine tetraaceticacid

Tc         tetracycline

TE         tris/ethylene diamine tetraacetic acid

TEM        transmission electron microscopy

TFP        type IV pilus

TIGR       the Institute for Genomic Research

TMHMM      transmembrane hidden Markov model

TPR        tetratrico peptide repeat

Tris       Tris[hydroxymethyl]aminomethane

tRNA       transfer ribonucleic acid

TRAP       tartrate-resistant acid phosphatase

TTSS       type III secretion system

USAMRIID   United States Army Medical Research Institute of

           Infectious Diseases

UV         ultra violet

VASP       vasodilator-stimulated phosphoprotein

wt         wild type




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Description: Molecular and cellular characterisation of Burkholderia ...