| Mycobacterium avium subsp. paratuberculosis (M. ap) is the causative agent of Johne's disease and is implicated in human Crohn's disease. To understand the molecular pathogenesis of this pathogen, we conducted genomic hybridization and transcriptomic profiling to identify genes that play roles in M. ap pathogenesis. Comparative genomic hybridizations of M. avium strains isolated from various host animals revealed a high level of genome plasticity among M. avium subspecies, especially those considered environmental strains. Generally, each subspecies in M. avium has unique genomic islands. In addition, three large inverted regions were identified between the M. avium 104 and M. ap K-10 genomes. On the other hand, genome variations are limited within M. ap strains isolated from ruminants and humans. We further identified a 644-kb inverted segment, spanning the origin of replication, between M. ap cattle and sheep strains. Analysis of the genome synteny suggests that the cattle strains could have differentiated the latest among the M. avium complex.;The global stress response of M. ap was defined by transcriptomic analysis. In general, responses to heat shock, acidity and oxidative stress were similar in M. ap and M. tuberculosis. Specifically, several sigma factors (e.g. sigH and sigE) were differentially co-regulated with a large number of genes depending on the type of each stressor. Additional groups of genes were regulated in the cow fecal samples.;Selected from the transcriptomic analysis, we subsequently analyzed the virulence of six M. ap mutants using a murine model of paratuberculosis. Bacterial and histopathological examinations indicated the attenuation of all mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Two of the mutants, DeltapstA and DeltagcpE, also showed reduced ability to invade calf intestine in a surgical model. Moreover, scanning electron microscopy, colonization levels and immunoblot analysis of the DeltapstA mutant indicated that pstA is also responsible for a novel cell wall lipopeptide, the ability to form biofilms and significant level of immunogenicity.;In summary, our study has gained insights into M. ap evolution and pathogenesis, and provides a knowledge base that will help with understanding M. ap-host interactions and vaccine development. |
