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

Nature Review Microbiology 2:189, 2004

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

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

Virulence factors in MAP Protein Phosphatase A Protein Kinase G

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

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

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

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

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

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

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

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

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

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

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

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

Antibodies against a-L5P Major cell wall-associated lipid Differs from MAA Eckstein et al. JBC 281:5209 2005

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

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

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