Survival tactics of M. paratuberculosis in bovine macrophage cells.

Mycobacterium paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium with the ability to survive and proliferate inside the vesicles of macrophage cells. How M. paratuberculosis and other mycobacteria survive in this hostile environment is not well understood. Present research findings can be divided into three possibilities: (1) mycobacteria may interfere with host protein expression and trafficking to stop vesicle maturation, (2) mycobacteria may express proteins that interfere with macrophage activation in a more direct manner, or (3) mycobacteria may enter macrophages in such a way as to avoid the normal process of activation via Toll-like receptors and other, as yet unknown mechanisms. Research thus far has predominately centered on possible macrophage/mycobacteria protein interactions. To more completely define how mycobacteria interfere with the process of phagosome maturation our group has recently taken a functional genomics approach, allowing the macrophage to "tell" us what host genes may be affected by phagocytosis of mycobacteria. We used DDRT-PCR to examine differences in macrophage cell gene expression during phagocytosis of E. coli and M. paratuberculosis. Macrophage cells not exposed to any phagocytosis target and in the process of phagocytosing latex beads were used as negative and positive controls, respectively. To date, we have identified 380 DDRT-PCR amplicons corresponding to transcripts whose expression profiles appear to be altered during the general process of phagocytosis. Dot-blot and Northern blot hybridizations with a subset of these amplicons were performed to confirm results observed with DDRT-PCR. Our preliminary results indicate that macrophage gene expression profiles change dramatically following phagocytosis and that gene expression profiles following phagocytosis of M. paratuberculosis are different than those following phagocytosis of E. coli or latex beads.