Towards the development of a rapid diagnostic test for by vev19514

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									Towards the development of
 a rapid diagnostic test for
Buruli ulcer: Identification of
  Mycobacterium ulcerans
      specific antigens
            Sacha J. Pidot, Jessica L. Porter
John K. Davies, Paul D. R. Johnson & Timothy P. Stinear
                 What do we want to achieve?




                                 Positive   Negative

www.mirates.nl
What do we want to achieve?




                        www.cellestis.com
                  Project aims

•    To develop immunodiagnostic tools for rapid
     identification of M. ulcerans (MU) infection
    – Identification of MU specific gene sequences
    – Cloning and overexpression of these sequences
    – Testing by Western blotting

•    Addresses a specific WHO Buruli Ulcer research
     priority
    M. ulcerans antigen identification
•    Need to identify antigens specific to
     M. ulcerans (MU)
•    Compare MU genome sequence to other
     mycobacterial genomes
    M. ulcerans antigen identification
•    Classes of MU specific
     proteins
    –   Membrane proteins
    –   Secreted proteins
    –   Hypothetical proteins
    –   Unique / Conserved
    –   Mycolactone PKS
        domains


•    Total of 41 potential
     antigens identified
 Determination of MU specific genes
• Are identified genes really MU
  specific?
• High genetic similarity between
  MU and M. marinum
• Sequenced M. marinum ‘M’ strain is lab
  adapted
• Are these genes present in non-sequenced
  isolates?
• Tested 27 diverse M. marinum strains for
  presence of MU genes by PCR
      Determination of MU specific genes
 Certain genes found in >90% of tested M. marinum
   strains - deletions in sequenced ‘M’ strain
                   Percentage of M. marinum strains containing MU genes

100

 90

 80

 70

 60

 50

 40

 30

 20

 10

  0
      MU29372   MU74349   MU520667   MU538935   MU540676   MU1214321 MU2891760 MU3579607 MU4686564 MU4690164
 Determination of MU specific genes

• 10 genes found to be present in tested
  M. marinum strains
• Removed non-MU specific genes from list
  of potential antigens
• Total of 31 potential antigens
• Do all MU strains contain these 31 genes?
• Tested 14 diverse MU strains by PCR
                Variable distribution of MU
                specific genes across strains
                Percentage of MU strains containing MU unique genes (14 strains)

90

80

70

60

50

40

30

20

10

0
                                                                         0


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Expression of recombinant antigens


              + inducer

                             Lysis

           Purification
     Reactivity of human sera against
               MU antigens
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                                                                                                                                        MUP15



                                                                                                                                                          Ag85A
                                                                                                                                                  MUP45
                                                                                                MUP45




                                                                                                                        MUP45
                                                                     MUP45
                                                             MUP15




                                                                                                                MUP15
                                                                                Ag85A




                                                                                                                                Ag85A
                                                                                        MUP15



                                                                                                        Ag85A
                                        Agy99
                        Agy99
      Agy99




                                                Chant
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64
                                                        64
50
                                                        50
36
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22
                                                        22
                      Antigens tested so far
 • 19 proteins tested so far - no serum reactivity to
   recombinant proteins observed to date
MUP13       Possible conserved membrane protein            Dehydratase                  PKS   domain
MUP14       Putative integral membrane protein             Ketoreductase A              PKS   domain
MUP15       Possible secreted protein                      Ketoreductase B              PKS   domain
MUP17       Possible conserved transmembrane protein       Acyltransferase acetate 1    PKS   domain
MUP45       Probable beta-ketoacyl synthase-like protein   Acyltransferase acetate 2    PKS   domain
MUP57       Possible lipoprotein                           Acyltransferase propionate   PKS   domain
MUP64       Possible conserved membrane protein            Enoylreductase               PKS   domain
MUP68       Conserved membrane protein                     Acyl Carrier Protein 1       PKS   domain
MUP74       Possible membrane protein                      Acyl Carrier Protein 2       PKS   domain
MUP76       Possible membrane protein                      Acyl Carrier Protein 3       PKS   domain
MU527240    Unique hypothetical protein                    Ketosynthase core            PKS   domain
MU527247    Unique hypothetical protein                    Ketosynthase alternate       PKS   domain
MU530424    Unique hypothetical protein                    hsp65           GroEL2
MU1050519   Unique hypothetical protein
MU1050554   Conserved membrane protein
MU1052616   Unique hypothetical membrane protein
MU3589715   Zinc metalloprotease
Ag85A       Part of known M.tb antigen complex
                 Future Directions
• Proteomic approach
• 2D gel electrophoresis and Western blotting
• Find proteins specifically
  recognised by patient sera,
  then identify by Mass
  Spectroscopy
               T-cell antigens
•   T-cells - alternate arm of immune system
•   Destroy infected cells
•   Recognise antigens presented by infected
    cells
•   Important in immunity to
    other mycobacterial
    diseases (TB, leprosy)
•   Are any of these antigens
    T-cell stimulators?
                 T-cell antigens

• Current case-control
  study in Victoria
   – Obtain patient blood
   – Separate PBMCs
   – Stimulate using
     purified MU antigens
   – Cytokine production /
     Proliferation
            Acknowledgements
•   Davies Lab
•   Tim Stinear & John Davies
•   May Smith, Travis Gooding, Frances
    Oppedisano – Royal Childrens’ Hospital,
    Victoria, Australia
     M




                                                                                              100




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     M
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  U         6
    52
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    53
M      04
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   10
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M        51
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M
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M        61
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M        43
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           5
                                                                                                    specific genes across strains
                                                                                                    Variable distribution of MU




                                                           % of strains
                                    % of african strains

								
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