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

Abstract

The Mycobacterium avium complex (MAC) is an important group of opportunistic pathogens for birds, cattle, swine, and immunosuppressed humans. Although invasion of epithelial cells lining the intestine is the chief point of entry for these organisms, little is known about the mechanisms by which members of the MAC are taken up by these cells. Studies with M. avium have shown that cytoskeletal rearrangement via activation of the small G-protein Cdc42 is involved and that this activation is regulated in part by the M. avium fadD2 gene. The fadD2 gene indirectly regulates a number of genes upon exposure to HEp-2 cells, including transcriptional regulators, membrane proteins, and secreted proteins. Overexpression of two fadD2-associated regulators (MAV_5138 and MAV_3679) led to increased invasion of HEp-2 cells, as well as altered expression of other genes. The protein product of one of the regulated genes, named CipA, has domains that resemble the PXXP motif of human Piccolo proteins, which bind SH3 domains in proteins involved in the scaffold complex formed during cytoskeletal rearrangement. Although CipA was not detected in the cytoplasm of HEp-2 cells exposed to M. avium, the recombinant protein was shown to be potentially expressed on the surface of Mycobacterium smegmatis incubated with HEp-2 cells and, possibly, to interact with human Cdc42. The interaction was then confirmed by showing that CipA activates Cdc42. These results suggest that members of the M. avium complex have a novel mechanism for activating cytoskeletal rearrangement, prompting uptake by host epithelial cells, and that this mechanism is regulated in part by fadD2, MAV_5138, and MAV_3679.

Internalization-dependent recognition ofMycobacterium aviumssp.paratuberculosisby intestinal epithelial cells

“…Further analysis indicated bacteria‐induced epithelial internalization through bacterial surface membrane molecules and active interference with cellular traffic (Bannantine et al. , 2003; Harriff et al. , 2009).…”

“…Using the highly specific clostridial toxin B serotype F we clearly demonstrate that bacterial uptake by small intestinal epithelial cells depends on the small GTPase Rac potentially in combination with Cdc42 (Alonso‐Hearn et al. , 2008; Harriff et al. , 2009).…”

Identification of Mycobacterium avium genes expressed during in vivo infection and the role of the oligopeptide transporter OppA in virulence

“…The Bacteriomatch II two-hybrid system (Stratagene, La Jolla CA) was used to construct and co-transform the bait pBT: opp A and the pray pTRG:MAC-library [17] into the reporter strain of E. coli . Transformants were plated onto LB agar plates containing 400 g/ml carbenicillin, 12.5 g/ml tetracycline, 34.5 g/ml chloramphenicol, and 50 g/ml kanamycin and incubated at 30°C overnight.…”

Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

“…The main route of infection in AIDS patients is the invasion of mucosal epithelial cells of the gastrointestinal tract, while in non-AIDS patients infections mainly occur through the respiratory route [9]. Recognition of M. avium by mouse macrophages involves binding of a 20 – 25 kDa lipoprotein from the cell envelope of M. avium to TLR2.…”