- Mycobacterium avium subsp hominissuis (MAH) is an opportunistic environmental pathogen that causes respiratory and gastrointestinal illness in immunocompromised persons such as those with chronic respiratory diseases or AIDs, respectively. In recent years, there has been a significant increase in the incidence of nontuberculous mycobacterial (NTM) lung infections, including in cystic fibrosis patients where MAH accounts for 72% of mycobacterial infections. Currently, there is no efficient approach to prevent or treat MAH infection in the lungs. During initial colonization of the airways, MAH form microaggregates composed of 3 to 20 bacteria on the human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. In this dissertation, the aims are to identify and characterize genes that are important for the invasive microaggregate phenotype and characterize the host response to microaggregate infection to further understand the early stages of MAH respiratory infection.
In Chapter 2, DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate- binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate
￼￼formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP- 1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an anti-vimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice.
In Chapter 3, the small hypothetical gene MAV_0831 (Microaggregate Invasion Protein-1, MIP-1) was identified as being upregulated during microaggregate formation. When MIP-1 was overexpressed in poorly-invasive M. smegmatis, it provided the bacterium the ability to bind and enter epithelial cells. In addition, incubating microaggregates with recombinant MIP-1 protein enhanced the ability of microaggregates to invade HEp-2 cells, and exposure to anti-MIP-1 immune serum reduced the invasion of the host epithelium. Through protein-protein interaction assays, MIP-1 was found to bind to the host protein filamin A, a cytoskeletal actin-binding protein integral to the modulation of host cell shape and migration. As visualized by immunofluorescence, filamin A was able to co-localize with microaggregates and to a lesser extent planktonic bacteria. Invasion of HEp-2 cells by microaggregates and planktonic bacteria was also inhibited by the addition of anti-filamin A antibody suggesting that filamin A plays an important role during infection. In addition, at earlier time points binding and invasion assay results suggest that MBP-1 participates significantly during the first interactions with the host cell while MIP-1 becomes important once the bacteria adhere to the host epithelium.
While these studies are valuable in understanding the pathogenesis of MAH, they primarily investigated the bacteria during microaggregate infection without commenting on the differences in the host response to microaggregate and planktonic infection. In Chapter 4, the bacteria-host interaction between microaggregates and
epithelial cells was examined in a variety of assays. Using a transwell polarized epithelial cell model, microaggregates tended to translocate through the monolayer more efficiently than planktonic bacteria. In addition, during infection with microaggregate and planktonic bacteria, host phosphorylated proteins were identified revealing differences in immune response, glutathione synthesis, and apoptosis. The host immune response was further investigated by measuring pro-inflammatory cytokine secretion during microaggregate and planktonic infection of BEAS-2B bronchial epithelial cells. The epithelial cells secreted more CCL5 during microaggregate infection suggesting that this chemokine may play an important role during microaggregate invasion. Collectively, this study confirms the different nature of infection by planktonic bacteria and microaggregates.
Overall, our data provide insights into the initial interaction between MAH and the respiratory mucosa. We characterized a pathogenic mechanism of infection utilized by MAH to manipulate the host respiratory epithelium and suggest new potential targets for the development of anti-virulence therapy.