Partial proteome of Mycobacterium avium subsp. paratuberculosis under oxidative and nitrosative stress

The growth pattern and protein expression profiles of sheep (S) and cattle (C) strains of Mycobacterium avium subsp. paratuberculosis (MAP) under oxidative and nitrosative stress were characterised. Oxidative stress was induced using 0.05% (v/v) H2O2 in BACTEC medium, and was lethal for an inoculum of 104 cells. However, an inoculum of 107 cells survived and proteomic changes were observed at 7 days. Nitrosative stress was induced using 1 mM NaNO2; it slowed the growth of an inoculum of 104 cells, but both strains recovered quickly when resuscitated in fresh media. Silver staining showed higher sensitivity for detection of 2D spots compared to SYPRO Ruby staining. A total of 18 proteins were regulated under oxidative and/or nitrosative stress. The expression of four antioxidant enzymes (AhpC, AhpD, OxcA and SodA) and four proteins involved in fatty acid metabolism (DesA2, FadA6_3, FabG and FadE19) was altered, together with a range of other proteins. Only one protein, AhpC was differentially regulated in both strains of MAP. Seven proteins (DesA2, AhpC, AhpD, Ppa, FabG, and hypothetical proteins MAP2411 and MAP 1885c) were identified in previous in vitro studies with temperature, hypoxia and/or nutrient starvation stressors and may be general stress response proteins of MAP. Prior studies have identified immune responses directed against AhpC and Ppa in animals with Johne's disease, expression of sodA and ppa within macrophages, and reduced virulence of impA mutants in mice, highlighting the relevance of proteomic studies using these in vitro stress models for pathogenesis studies.