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

Abstract

SummaryMycobacterium 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 infection through the gut, binding and invasion of the intestinal epithelial barrier are required. To characterize this process further, we determined the cell surface(s) (basolateral vs. apical membrane) that M. avium interacts with 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 similar when the assay was performed with control medium in the presence of Ca 21 (when only the apical surface was exposed), with Ca 21 -depleted medium or with Ca 21 -depleted medium 1 1 mM EGTA (exposure of both apical and basolateral membranes), suggesting that the bacterium enters the apical surface of the epithelial lining. These observations were confirmed by assays in a transwell system and by using fluorescent microscopy. Real-time video microscopy showed that M. avium entry was not associated with membrane ruffling and the use of pharmacological inhibitors of the small GTPases demonstrated that 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 significantly greater (between five-and ninefold) ability to bind to and invade new monolayers of epithelial cells or macrophages when compared with the invasion by M. avium grown on agar (extracellular growth; EG). IG phenotype 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 within the cells, changes phenotype, becoming more invasive towards both macrophages and other epithelial cells.

Identification of Mycobacterium avium Genes That Affect Invasion of the Intestinal Epithelium

“…M. avium binding and invasion of epithelial intestinal cells in vivo appear to require an array of moieties to interact with mucin, microvilli, and membranes of the host cell, triggering cytoskeletal reorganization (9), activation of RhoA (35), and protein phosphorylation (9).…”

“…The bacterium can resist acidic conditions in the stomach and colonize the intestinal tract (10,23). Recent studies have demonstrated that M. avium interacts with the intestinal mucosa, entering primarily enterocytes (7,35). Invasion of enterocytes appears to occur through the apical membrane rather than the basolateral surface (35).…”

“…Recent studies have demonstrated that M. avium interacts with the intestinal mucosa, entering primarily enterocytes (7,35). Invasion of enterocytes appears to occur through the apical membrane rather than the basolateral surface (35). Therefore, it is likely that the abilities to invade and translocate across the intestinal mucosa are important components of M. avium pathogenesis.…”

Mycobacterium aviumGenes MAV_5138 and MAV_3679 Are Transcriptional Regulators That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a Putative Surface Protein Interacting with Host Cell Signaling Pathways

“…Assays were performed as described previously by Sangari et al (31). Briefly, MAC109, MAC109:MAV_5138, and MAC109: MAV_3579 (two clones overexpressing the transcription regulator [see Table 2]) were adjusted to 10 8 /ml by McFarland standards and verified by plating serial dilutions.…”

“…Kitaura et al (20) found five M. avium proteins that bind fibronectin, including Ag85 and Mpb51. Fibronectin is expressed on the surface of M cells rather than enterocytes, while M. avium preferentially enters enterocytes (31), suggesting that these proteins are not primarily important for epithelial cell invasion. A recent study identifying secreted proteins of M. tuberculosis by proteomic methods indicated that a large portion of the secreted proteins were previously unknown and that almost 40% of the proteins were secreted by a mechanism other than the general secretory pathway (23), indicating there are also likely to be many surface and secreted proteins and systems by which these proteins are secreted by M. avium that are not yet identified.…”