Antigen expression and metabolism of Mycobacterium avium subsp. paratuberculosis in vivo

Bovine Johne’s disease (paratuberculosis), caused by Mycobacterium avium subspecies paratuberculosis (MAP), poses a significant economic problem to the beef and dairy industry worldwide. Also, MAP has been implicated to play a role in some cases of Crohn’s disease (CD), type 1 diabetes mellitus and multiple sclerosis in humans. Furthermore, MAP diagnostics are hampered by a lack of assays with high diagnostic sensitivity and specificity, virulence determinants and metabolism of MAP are mostly unexplored, and the pathogenesis of Johne’s disease (JD) is only partially resolved. Since mycobacterial membrane proteins expressed during infection are likely to play an important role in pathogenesis, membrane-enriched fractions, namely mucosa-derived membranes (MDM) and culture-derived membranes (CDM), of M. avium subsp. paratuberculosis from three cows with clinical paratuberculosis were investigated. In this work 48 predicted membrane proteins were identified, 13 of which have been previously associated with mycobacterial virulence (AtpF, AtpH, LprG, HBHA, SenX3, Mce, CipA, MAP2121c, MAP3305c) or the virulence of other intracellular pathogens (PepA, HtrA, SecE, MviN). Four membrane proteins (HBHA/MAP3968c, MAP0010c, MAP1775, MAP2643) and three possible membrane-associated proteins (MAP1205, MAP2057, MAP3933c) revealed higher expression in MDM compared to CDM and might play a specific role in virulence of MAP. Comparative investigation of cytoplasmic fractions (MDC vs. CDC) using high-throughput mass spectrometry (LC-MS/MS) in combination with quantitative 2D gelectrophoresis (2D DIGE) facilitated a prediction of the MAP metabolome in its natural host; predicted enzymatic functions were based on current knowledge of the Mycobacterium tuberculosis (MTB) metabolome in culture. Our results showed that, at large, the central metabolism of MAP in the host differs only slightly from that in culture. It appeared to be driven by β-oxidation of lipids, most probable host cell cholesterol. Beta-oxidation is accompanied by an ...