Abstract
Antibodies play a central role in host immunity by directly inactivating or recognizing an invading pathogen to enhance different immune responses to combat the invader. However, the cellular responses of pathogens to the presence of antibodies are not well-characterized. Here, we used different mass spectrometry techniques to study the cellular responses of the pathogenic fungus Histoplasma capsulatum to monoclonal antibodies (mAb) against HSP60, the surface protein involved in infection. A proteomic analysis of H. capsulatum yeast cells revealed that mAb binding regulates a variety of metabolic and signaling pathways, including fatty acid metabolism, sterol metabolism, MAPK signaling and ubiquitin-mediated proteolysis. The regulation of the fatty acid metabolism was accompanied by increases in the level of polyunsaturated fatty acids, which further augmented the degree of unsaturated lipids in H. capsulatum’s membranes and energy storage lipids, such as triacylglycerols, phosphatidylcholines, phosphatidylethanolamines and phosphatidylinositols. MAb treatment also regulated sterol metabolism by increasing the levels of cholesterol and ergosterol in the cells. We also showed that global changes in the lipid profiles resulted in an increased susceptibility of H. capsulatum to the ergosterol-targeting drug amphotericin B. Overall, our data showed that mAb induction of global changes in the composition of H. capsulatum membranes can potentially impact antifungal treatment during histoplasmosis.
Highlights
Antibodies are major components of the humoral immune response and primarily fight infections by facilitating phagocytosis, activating the complement system, neutralizing or lysing pathogens or mediating specific cytotoxic responses [1]
Control samples of H. capsulatum were prepared in parallel, one treated with phosphate-buffered saline (PBS) and the other treated with the same hybridoma media used to produce the monoclonal antibodies (mAb)
Treated cells were submitted to a simultaneous metabolite, protein and lipid extraction (MPLEx) consisting of the extracted proteins digested with trypsin and the resulting peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Summary
Antibodies are major components of the humoral immune response and primarily fight infections by facilitating phagocytosis (opsonization), activating the complement system, neutralizing or lysing pathogens or mediating specific cytotoxic responses [1]. Antibodies against components of fungal cells, such as capsular polysaccharide, β-glucans, glycolipids, melanin and proteins, can have protective roles during infections. Antibodies against glucoroxylomann (GXM), the main component of Cryptococcus neoformans capsule, inhibit the release of polysaccharide from the fungus [2] and hydrolyze glycans and peptides [3]. Other antibodies, such as an anti-monohexosylceramide antibody, have growth-inhibitory activity on C. neoformans [4]. Engineered chimeric antibodies have been shown to confer pan-fungal protection by targeting chitin, a conserved polysaccharide present across the Fungi kingdom [5].
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