Graduation date: 2008
Environmental mycobacteria are important opportunistic pathogens for many hosts,
including humans, cattle, and fish. Two well-studied species are Mycobacterium
avium subsp. avium, a significant cause of disseminated bacterial disease in patients
with AIDS, and Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s
disease in cattle. Many other species that are considerable sources of infections in
fish, such as Mycobacterium chelonae and Mycobacterium marinum, also have
zoonotic potential. To gain knowledge about the invasion of epithelial cells by
environmental mycobacteria, selected genes and proteins involved in the uptake of M.
avium by HEp-2 cells were analyzed by a variety of methods. Two transcriptional
regulators (MAV_3679 and MAV_5138) were identified as being involved in
invasion. A mycobacterial protein (CipA) with an amino acid sequence suggestive of
an ability to be a part of the scaffolding complex that forms during cell signaling
leading to actin polymerization was found to putatively interact with host cell Cdc42.
Fusion of CipA to GFP, expressed in Mycobacterium smegmatis, revealed that CipA
localizes to a structure on the surface of bacteria approaching HEp-2 cells. To
establish whether species of environmental mycobacteria isolated from different hosts
use similar mechanisms to M. avium for interaction with the mucosa, and for survival
in macrophages, assays to determine invasion and replication were performed in
different cell types, and a custom DNA microarray containing probes for known
mycobacterial virulence determinants was developed. Bacteria cultured from
macrophages indicated differences between the ability of M. avium and other
environmental species to replicate in human, mouse, and carp cell lines.
Hybridization of genomic DNA isolated from EM species against the sequenced
MAC104 strain of M. avium showed that there are genes and regions of the
chromosome absent from a subset of those species that may be important in
determining host specificity and the in differences in virulence observed in vitro and
in vivo. Finally, the intestinal epithelium was determined to be the primary route of
infection for mycobacteria in zebrafish, similar to M. avium infection in humans. M.
avium does not infect zebrafish, but the comparative virulence and genomics of some
species isolated from fish suggest that they could be used as a surrogate model to
continue progress in understanding the interaction between environmental
mycobacteria and epithelial cells.