Inhibition of phagosome maturation and survival of Mycobacterium avium subspecies paratuberculosis in polymorphonuclear leukocytes from Crohn's disease patients.

摘要

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that is known to parasitize macrophages. MAP is the known etiological agent of Johne's disease and implicated in the etiology of Crohn's disease. MATERIAL/METHODS: In this study, the survival of human-derived MAP isolate following phagocytosis was evaluated using murine macrophage cell line J774A.1 and polymorphonuclear cells (PMNC's) from six Crohn's disease patients. PMNC's from five healthy individuals and four ulcerative colitis patients, as well as Escherichia coli and Mycobacterium tuberculosis, were included as controls (MOI 10:1). Maturation of the phagosome was determined by evaluating the presence of stage specific markers on the surface of the phagosomal membrane. The endosomal protein, transferrin receptor, and the lysosomal protein, Lamp-1, were then immunostained with Cy-5 conjugated secondary antibodies, and colocalization of bacteria with each marker was evaluated separately using confocal scanning laser microscopy (CSLM). RESULTS: In both models, colocalization of viable MAP and M. tuberculosis with the early endosomal marker occurred with a higher frequency than did association with the late lysosomal marker, as compared to live E. coli, and all dead bacterial species. Using differential live/dead staining and fluorescent microscopy, survival of M. tuberculosis and MAP was calculated to be 85% and 79%, respectively compared to only 14% for E. coli. CONCLUSIONS: Overall, MAP survival in murine macrophages and human PMNCs appears to mimic M. tuberculosis, suggesting the ability of this microorganism to resist phagolysosome fusion, by maintaining association with the early endosomes. The data supports MAP virulence in humans.