Understanding Mycobacterium avium subspecies hominissuis microaggregate mediated pathogenesis

Abstract

Mycobacterium avium subspecies hominissuis (MAH) is an opportunistic pathogen and causes nontuberculous infections in immune compromised individuals, an emerging problem that has been recognized worldwide. Understanding the pathogenesis of this organism is important as better treatment and prevention options are needed. Microaggregates form when two or more bacterial cells join at a surface. MAH forms micgroaggregates to promote its entry in to epithelial cells and cause infection. The mechanisms involved in the interaction between the microaggregate and the host are becoming clearer as the molecules involved in this process are being uncovered. Microaggregate Invasion Protein-1 (MIP-1) is now described as having a major role in the invasion of epithelial cells by MAH.

Mycobacterium avium subspecies hominissuis (MAH) is a member of the Mycobacterium avium complex, characterized as a subspecies that infects both humans and swine.¹ MAH is an opportunistic pathogen and ubiquitous in the environment. In humans, MAH can cause nontuberculous infections in immune compromised individuals; a problem that is becoming increasingly recognized worldwide.^2,3 These infections are mainly of the lung but may also be systemic or of other body sites. Therapy can be a challenge for patients with nontuberculous infections as treatment is lengthy, it requires multiple antibiotics, and there is a high incidence of re-occurrence.² In animal health, MAH is mainly a problem in the swine industry where infection leads to lesions found at meat inspection that are indistinguishable from those caused by Mycobacterium bovis, leading to meat condemnation. The zoonotic potential of MAH has not been ruled out, also adding to the importance of this pathogen.⁴

A central part of the pathogenesis of MAH is its entry in to the host cell. For this both attachment and invasion must occur. Research has shown that entry in to epithelial cells by Mycobacterium avium occurs more efficiently when it is able to aggregate and form biofilm.⁵ A microaggregate is the association of 2 or more bacteria which attach to a surface often leading to the formation of biofilm. Many bacteria aggregate and form biofilm to allow for infection including Pseudomonas aeruginosa,⁶ Streptococcus pyogenes,⁷ and Neisseria meningitides.⁸ This process has been observed to be mediated by different bacterial components including type IV pili, flagella and type III secretion system (Pseudomonas aeruginosa)^6,9,10 or surface exposed proteins (Streptococcus pyogenes).⁷

In this issue of Virulence, Babrak et al.¹¹ have discovered an additional mechanism that mediates the initial interaction of MAH and the epithelial cell in microaggregates. A previous study by the same research group described Microaggregate Binding Protein-1, which is involved in adhesion to epithelial cells.¹² Microarray gene expression data of that study also pointed to the up-regulation of the hypothetical gene MAV_3013 during microaggregate formation.¹² Babrak et al. (2015)¹¹ have characterized MAV_3013 as Microaggregate Invasion Protein-1 (MIP-1) demonstrating its role in infection and invasion of cells.

MIP-1 was found to be involved in the invasion mediated by MAH as a result of different experiments. Firstly, it was made clear that the protein was involved in invasion of epithelial cells using Mycobacterium smegmatis as a model. Mycobacterium smegmatis is a bacterium that invades epithelial cells poorly and when the over-expression of MIP-1 was induced there was a significant increase in both binding and invasion compared to a strain not expressing the protein. Further evidence to show that MIP-1 is involved in microaggregate invasion was obtained when analyzing the effect of blocking MIP-1. When MAH microaggregates were incubated with anti MIP-1 immune serum, a significant reduction in invasion of HEp-2 cells was observed. To assess if this interaction occurred in vivo, mice were infected with MAH microaggregates that had been previously incubated with anti-MIP-1 immune serum. The incubation with anti-MIP-1 antibodies led to a significant reduction of MAH recovered from the lungs of mice as compared to those that received microaggregates alone, thus confirming MIP-1 as an important protein involved in MAH pathogenesis and invasion.

In this study, the ligand of MIP-1 was also discovered. By far-Western blot, a lysate of HEp-2 was run on a gel and transferred to a nitrocellulose membrane which was then probed with MIP-1. The strongest band was then excised and submitted to mass spectrometry. Filamin A was identified as the major protein interacting with MIP-1. Filamin A is an actin binding protein of the filamin family, which are multi-functional proteins that mediate positioning of the cytoskeleton.¹³ The function of filamin A gives clues as to how MIP-1 mediates invasion since it is involved in cellular movement. To confirm filamin A as the host cell ligand, HEp-2 cells were incubated with anti-filamin A antibody to block the protein prior to invasion assays. This treatment led to significant reduction in invasion of bacteria, confirming the role of filamin A in this process. Additionally, both MIP-1 and filamin A were found to co-localize by immunofluorescence staining.

As the model currently stands, it seems as though MBP-1 is involved in the earlier processes of microaggregate formation, especially with binding to the host epithelial cell. Once bound, MAH aggregates at the cell surface, and through the action of MIP-1, is internalized by the host. Additional proteins may also be involved in microaggregate formation and invasion and it remains to be discovered which cues from the host and/or bacterium stimulate microaggregate formation.

This study is a good example of how gene expression data can be followed up to gain meaningful insight in to pathogenesis. The MIP-1 protein was characterized along with its role in invasion. Its ligand, filamin A, was also uncovered. Additional research using cell lines other than HEp-2 (a human laryngeal epithelial cell line) would be interesting to confirm that MIP-1 is involved in invasion of other cell types, including those of swine. Perhaps in the future it would also be relevant to investigate the role of MIP-1 and MBP-1 in biofilm formation and if it occurs in their absence.

Considering the difficulty of treating MAH infections, insights in to how infection is established are crucial for the development of novel therapeutic strategies. The finding that blocking MIP-1 leads to significant reduction in colonization of the mouse lung gives hope for the development of novel treatments. Research should be carried forward to continue to understand microaggregate formation and function: this may hold the key for the development of novel therapies which are much needed for the treatment of MAH and nontuberculous infections.