Abstract
Pathogenic mycobacteria are important agents causing human disease. Mycobacterium avium subsp. hominissuis (M. avium) is a species of recalcitrant environmental pathogen. The bacterium forms robust biofilms that allow it to colonize and persist in austere environments, such as residential and commercial water systems. M. avium is also an opportunistic pathogen that is a significant source of mortality for immune-compromised individuals. Proteins exposed at the bacterial surface play a central role in mediating the relationship between the bacterium and its environment. The processes underlying both biofilm formation and pathogenesis are directly dependent on this essential subset of the bacterial proteome. Therefore, the characterization of the surface-exposed proteome is an important step towards an improved understanding of the mycobacterial biology and pathogenesis. Here we examined the complement of surface exposed proteins from Mycobacterium avium 104, a clinical isolate and reference strain of Mycobacterium avium subsp. hominissuis. To profile the surface-exposed proteins of viable M. avium 104, bacteria were covalently labeled with a membrane impermeable biotinylation reagent and labeled proteins were affinity purified via the biotin-streptavidin interaction. The results provide a helpful snapshot of the surface-exposed proteome of this frequently utilized reference strain of M. avium. A Cu-Zn SOD knockout mutant, MAV_2043, a surface identified protein, was evaluated regarding its role in the survival in both macrophages and neutrophils.
Highlights
Pathogenic mycobacteria are responsible for a large number of human infections
Preparation of M. avium cultures M. avium 104 was grown on Middlebrook 7H10 agar and transferred into 200 ml of Middlebrook 7H9 broth supplemented with 10% oleic albumin dextrose catalase (OADC) (Hardy Diagnostics, Santa Maria CA) and cultured at 37°C with constant agitation
Comparison of protein extraction and affinity purification buffers Prior to initiating this study, several potential buffer components were tested for their capacity to solubilize mycobacterial proteins and their compatibility with the streptavidin-biotin affinity interaction
Summary
Pathogenic mycobacteria are responsible for a large number of human infections. The interaction between the pathogen and the human host is complex, but surface-exposed molecules are very significant in several aspects of the interaction.An opportunistic pathogen, Mycobacterium avium subsp. hominissuis (M. avium) rose to prominence during the HIV/ AIDS epidemic of the 1980’s and 1990’s [1]. Pathogenic mycobacteria are responsible for a large number of human infections. The interaction between the pathogen and the human host is complex, but surface-exposed molecules are very significant in several aspects of the interaction. M. avium can survive and proliferate within the intra-cellular environment of host phagocytes, The surface-exposed proteome of a bacterium is of great interest to both microbiologists and immunologists. Surface-expressed proteins are likely to be important to interaction with phagocyte cells [5,6]. Surface proteins are primary targets for both innate and adaptive immune responses. Effective engagement of pathogens by the immune system requires the recognition of accessible targets, which tend to be surfaceexposed molecules. Surface-exposed proteins are disproportionately represented in the antigenic profiles from mycobacteria-infected animal hosts [7,8]
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