1. Virulence Factors as Potential Antigens for Serologic Tests Horacio Bach, Ph.D. Division of Infectious Diseases Department of Medicine University of British Columbia

2. Nature Review Microbiology 2:189, 2004

3. Hestvik et al., FEMS Microb.Rev 29:1041, 2005
ER = Endoplasmic reticulum
LAM = Lipoarabinomannan
PIM = Phosphatidylinositol mannoside
TGN = Trans-Golgi network
Hestvik et al., FEMS Microb.Rev 29:1041, 2005

4. Virulence factors Molecules secreted by the pathogen into the host
Block essential pathways to allow survival
Produced de novo for new cell infection
Candidates to be processed in the lysosome and
presented to antibody-producing cells

5. Virulence factors in MAP
Protein Phosphatase A
Protein Kinase G

6. Protein Phosphorylation Systems
Three systems in bacteria
Signal Transduction (modification of proteins)
1) “Two-component” system
Predominant in prokaryotes
Two proteins involved in signal transfer
2) Classical ATP/GTP-dependent system
Predominant in eukaryotes
O-kinases-phosphorylate OH groups of serine/threonine or tyrosine
Formation of phosphomonoesters
Dephosphorylated by specific O-phosphatases

7. Signal Transduction in Mycobacteria
11 Two component system
11 Eukaryotic serine-threonine kinases
2 Protein tyrosine phosphatases (PtpA and PtpB
1 Acid phosphatase (SapM)

8. Protein Phosphatase A
Dephosphorylates the macrophage substrate VPS33B, leading to phago-lysosome inhibition
Bach et al., Cell Host Microbe 3:316, 2008

9. Protein Phosphatase A Prevents phagosome acidification
Wong et al., PNAS, 108:19371, 2011

10. Protein Phosphatase A in MAP
Killed
24 h
48 h
72 h
Live
Map
PtpA
Bach et al., Infection & Immunity 74:6540, 2006

11. Protein Kinase G - PknG Soluble Ser/Thr kinase with endogenous and
exogenous activity
Secreted upon macrophage infection with BCG
Inhibit phago-lysosome fusion
Walburger et al., Science 304:1800, 2004

12. Analysis of PtpA and PknG in CD patients
Caco-2
THP-1
MAP isolates used were cow (squares), caprine (triangle), and rabbit (circle)
Bach et al., Scand J Gastro 46:30, 2010

13. Analysis of PtpA and PknG in CD patients
Sample used: serum
Frozen same day
Test: in-house ELISA
ELISA control: recombinant protein
Bach et al., Scand J Gastro 46:30, 2010

14. Analysis of PtpA and PknG in CD patients
Control
PtpA
0.174
0.120
PknG
0.198
0.135
Wilconox Rank Sum, p<0.01
Bach et al., Scand J Gastro 46:30, 2010

15. aPtpA antibodies in CD under treatment
Xia et al., Scand J Gastro 49:157, 2014

16. aPtpA antibodies in CD under treatment
Xia et al., Scand J Gastro 49:157, 2014

17. aPtpA antibodies in CD under treatment
A = Azathioprine
M = 5-ASA
S = Steroids
Xia et al., Scand J Gastro 49:157, 2014

18. Antibodies against a-L5P
Major cell wall-associated lipid
Differs from MAA
Eckstein et al. JBC 281:

19. Antibodies against a-L5P
Bacterium (Texas Red)
a-L5P (FITC)
MAP
MAA

20. University of British Columbia
Dr. Brian Bressler – Gastroenterology
Dr. Yossef Av-Gay – Infectious Diseases
Mount Sinai Hospital - Toronto
Dr. Mark Silverberg – Gastroenterology
Joanne Stempak– Gastroenterology
INRA- France
Dr. Jean Marc Reyrat
Dr. Frank Biet
McGill University
Dr. Marcel Behr

21. THANK YOU FOR YOUR ATTENTION