External pathogenic fungal proteome

H. capsulatum has evolved a number of responses to the signals produced by the host antimicrobial activities such as:  numerous catalase enzymes with antioxidant activities, an alternative oxidative phosphorylation pathway with metabolic and antioxidant activities,  and a complex pathway to mitigate the toxic conditions of the phagolysosome and a considerable redundancy in these responses only adds to their initial robustness during disease pathogenesis. A number of the components involved in these defensive responses are surface or externalized proteins such as the M antigen or catalase B enzyme  catalase A (the homolog of the N. crassa CAT1 gene), the product of the CBP1 gene, the homolog of the heat shock 70 protein, the C-18 ligand or the surface localized heat shock 60 protein (hsp60), and the product of the YPS3 gene. Others are found to increase after stress – for example, chitin synthase 2. The H. capsulatum catalase and heat shock proteins are known stress response proteins and these along with the other externalized proteins are either known virulence factors or likely involved in promoting the dimorphic transition and pathogenesis.

Pathogenesis of Histoplasma capsulatum and pulmonary mycobacteria share similarities in their ability to manipulate the host cell environment through a complex cascade of pathways involving initiating phagocytosis by the alveolar macrophage followed by the ability to grow and propagate in their hostile environment by inhibition of phagolysosome maturation resulting in its conversion into a hospitable “pathogen incubator”. Similarly, gram negative bacteria synthesize and secrete 50 to 100 nm outer membrane vesicles that while difficult to detect in host fluids have just recently been identified in host serum and urine samples. This work shows that the synthesis and externalization of these vesicles is part of a new secretion system (type VI), likely involved in the externalization of toxins and host modulating “effectors”, and is a pathogen virulence factor (for review see). More recently, results indicate a role for the endocytic/exocytic pathways in fungal pathogenesis. The C. albicans endosomal regulators CaVPS34 (PI3-kinase) and Vac1p (a vesicle transporter protein) were identified as virulence factors and in Cryptococcus neoformans small nanometer sized vesicles, containing the serum antigen and virulence factor GXM, are externalized through the cell membrane/wall structures. Vaccination with C. neoformans cell free growth media or a H. capsulatum surface derived antigen preparation elicits full or partial protection against histoplasmosis in mice.

Collectively, these results support the proposals that many pathogens produce and externalize “effectors” to modulate and mitigate the host antimicrobial response at numerous levels. Therefore, we initiated a careful characterization of the H. capsulatum “external proteome”. Using 2DE and mass spectrometry analysis we resolved 2 subproteomes from the H. capsulatum external proteome (a component proteome of the externalized proteins of the H. capsulatum functional secretome).  These results show that many of these proteome components are in multiple highly modified and isomeric forms. Initial analysis, by mass spectrometry, shows that these proteomes are likely comprised of thousands of proteins that are involved in numerous complex pathways. As well, one of these subproteomes copurifies with small nano-vesicles (exosomes/pathosomes) and therefore, imply an enocytic/exocitic pathway involvment in their externalization similar to that found in these other systems. Finally, we find that this external proteome, when used as a vaccine in mice, results in significant reductions in the yeast load in lung and spleen tissue.