Mycobacterium avium enters intestinal epithelial cells through the apical membrane, but not by the basolateral surface, activates small GTPase Rho and, once within epithelial cells, expresses an invasive phenotype

Mycobacterium avium is a common pathogen in AIDS patients that is primarily (but not exclusively) acquired through the gastrointestinal tract, leading to the development of bacteraemia and disseminated disease. To cause infec tion through the gut, binding and invasion of the intestinal epithelial bar rier are required, To characterize this process further, we determined the cell surface(s) (basolateral vs, apical membrane) that M. avium interacts w ith in intestinal mucosal cells in vitro. The level of binding and invasion of both HT-29 and Caco-2 intestinal cell monolayers by M. avium were simil ar when the assay was performed with control medium in the presence of Ca2 (when only the apical surface was exposed), with Ca2+-depleted medium or w ith Ca2+-depleted medium + 1 mM EGTA (exposure of both apical and basolater al membranes), suggesting that the bacterium enters the apical surface of t he epithelial lining, These observations were confirmed by assays in a tran swell system and by using fluorescent microscopy, Real-time video microscop y showed that M, avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated tha t M. avium invasion is dependent on the activation of the small GTPases Rho , but not on Rac or Cdc42, Passage of M. avium through HT-29 cells led to a phenotypic change (intracellular growth; IG) that was associated with a si gnificantly greater (between five- and ninefold) ability to bind to and inv ade new monolayers of epithelial cells or macrophages when compared with th e invasion by M. avium grown on agar (extracellular growth; EG), IG phenoty pe invasion of HT-29 cells also takes place only by the apical surface. M. avium enters intestinal epithelial cells by the apical surface and, once wi thin the cells, changes phenotype, becoming more invasive towards both macr ophages and other epithelial cells.