Characterization of the M antigen of Histoplasma capsulatum: Function and expression

The dimorphic fungal pathogen Histoplasma capsulatum produces an immunodominant secreted glycoprotein known as the M antigen. We characterized the M antigen/secreted catalase, performed functional analyses, and addressed its role in virulence. Notable differences in secreted catalase activity were observed among H. capsulatum strains belonging to three different restriction fragment length polymorphism (RFLP) classes; RFLP class III strains displayed high levels of secreted catalase activity whereas RFLP class I and II strains displayed lower levels of activity. We overexpressed CATB (M antigen) in H. capsulatum from a multicopy telomeric plasmid, as demonstrated by higher CATB mRNA levels in Northern blots. Overexpression of CATB (M antigen) correlated with increased secreted catalase activity, providing direct experimental support for the function of M antigen as a catalase. We next examined the effect of secreted catalase overexpression on resistance to H₂O₂ and on the virulence of H. capsulatum in macrophage virulence assays and in a mouse model of infection. Overexpression of the secreted catalase did not increase H. capsulatum virulence in any of the infection models and did not significantly affect H₂O₂ sensitivity. We also examined the expression of H. capsulatum CATB under environmental conditions known to influence catalase expression in other organisms and/or stimuli that the fungus might encounter during infection and host responses. Our results demonstrate control at the RNA level of H. capsulatum CATB (M antigen) under a variety of growth conditions. Notably, H. capsulatum infection of either non-activated or IFNγ plus LPS activated RAW 264.7 macrophages resulted in CATB down-regulation when compared to H. capsulatum grown in vitro. Collectively, this work has provided functional evidence that the M antigen of the dimorphic fungal pathogen H. capsulatum is a catalase and has identified environmental conditions that affect CATB (M antigen) expression. Additionally, it provided evidence consistent with lack of a contribution of M antigen to H. capsulatum virulence. However, it will be necessary to generate and examine virulence of isogenic H. capsulatum strains deficient in M antigen/catalase production as well as complemented strains to definitively address the role of the M antigen/secreted catalase in H. capsulatum pathogenesis, by fulfilling molecular Koch's postulates.