The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells

Abstract

"Mycobacterium avium subsp. hominissuis" is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregatebinding 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 antivimentin 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. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy. " M ycobacterium avium subsp. hominissuis" is an environmental bacterium ubiquitous in soil and natural water sources. In addition, M. avium subsp. hominissuis is an opportunistic pathogen that poses a major risk to immunocompromised patients, such as those with HIV infection, cystic fibrosis, and chronic respiratory diseases such as bronchiectasis and chronic obstructive pulmonary diseases (COPD). During the AIDS epidemic, mycobacterial infections became a widespread medical concern due to high morbidity and mortality in severely immunocompromised individuals (1). In recent years, there has been a significant increase in the incidence of nontuberculous mycobacterial (NTM) lung infections, including in cystic fibrosis patients, where M. avium subsp. hominissuis accounts for 72% of mycobacterial infections (2-4). Studies have also found an increase in NTM lung infections in middle-aged women with no known underlying conditions (5).Due to the hardy cell wall of M. avium subsp. hominissuis and its natural resistance to many antibiotics, treatment is lengthy and encompasses a combination of various antibiotics, such as macrolides and ethambut...

Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

“…Typhimurium (Diard et al, 2013), and a variety of mycobacterial species are reported to readily alter their phenotypes in response to external stimuli. The close relative of MAP, M. avium subspecies hominissuis, alters its phenotype upon the formation of microaggregates and biofilms (Babrak et al, 2015;McNabe et al, 2011), in response to the metal matrix found within host phagosomes (Early & Bermudez, 2011) and upon intracellular infection of macrophages and amoebae (Bermudez et al, 2004;Harriff & Bermudez, 2009 Patel et al, 2006). Our data suggest that MAP changes its phenotype during the stages of intracellular infection, and that these changes involve the alteration of whole cell-and cell wall-associated lipids.…”

Microaggregate-associated protein involved in invasion of epithelial cells byMycobacterium aviumsubsp.hominissuis

“…17 Yamazaki et al, Babrak et al 2015, examined the initial interaction between MAH and respiratory epithelial cells and identified an invasive phenotype coupled with microaggregate formation when the bacteria were incubated with respiratory BEAS-2B bronchial epithelial cells for 24 hours as well as in vivo in a mouse model. 17,18 Subsequently, we genetically characterized the invasive microaggregate phenotype through DNA microarray, identifying highly regulated genes during microaggregate formation. 17 The protein, MAV_3013 (MBP-1) was identified as a surface-exposed, small protein, responsible for microaggregate attachment to the host epithelium through the interaction with the host intermediate protein, vimentin.…”

“…16 After adhering to the respiratory mucosa, there are several mechanisms in which the bacteria can form microaggregates: bacteria can replicate forming clonal microaggregates or bacteria can secrete extracellular signals to initiate recruitment of bacteria to the site of microaggregate formation. 15,17 MAH microaggregates consist of actively recruited planktonic bacteria, although a chemotactic substance or protein responsible for aggregation has not been elucidated. 17 Yamazaki et al, Babrak et al 2015, examined the initial interaction between MAH and respiratory epithelial cells and identified an invasive phenotype coupled with microaggregate formation when the bacteria were incubated with respiratory BEAS-2B bronchial epithelial cells for 24 hours as well as in vivo in a mouse model.…”

“…15,17 MAH microaggregates consist of actively recruited planktonic bacteria, although a chemotactic substance or protein responsible for aggregation has not been elucidated. 17 Yamazaki et al, Babrak et al 2015, examined the initial interaction between MAH and respiratory epithelial cells and identified an invasive phenotype coupled with microaggregate formation when the bacteria were incubated with respiratory BEAS-2B bronchial epithelial cells for 24 hours as well as in vivo in a mouse model. 17,18 Subsequently, we genetically characterized the invasive microaggregate phenotype through DNA microarray, identifying highly regulated genes during microaggregate formation.…”

“…17,18 Subsequently, we genetically characterized the invasive microaggregate phenotype through DNA microarray, identifying highly regulated genes during microaggregate formation. 17 The protein, MAV_3013 (MBP-1) was identified as a surface-exposed, small protein, responsible for microaggregate attachment to the host epithelium through the interaction with the host intermediate protein, vimentin. MBP-1 utilizes the host protein vimentin as a surface exposed receptor for microaggregate binding to the epithelial cell wall.…”