The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the Histoplasma secreted proteome, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells suggesting its role in pathogenesis. As tools for characterization and detection of Cfp4, we have generated three monoclonal antibodies and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutage... More
The yeast phase of Histoplasma capsulatum is the virulent form of this thermally dimorphic fungal pathogen. Among the Histoplasma secreted proteome, culture filtrate protein 4 (Cfp4) is a heavily glycosylated factor produced abundantly and specifically by Histoplasma yeast cells suggesting its role in pathogenesis. As tools for characterization and detection of Cfp4, we have generated three monoclonal antibodies and determined the epitope each recognizes. Through site-directed mutagenesis of Cfp4, we identified three asparagines that function as the principal sites of N-linked glycan modification. To test the function of Cfp4 in Histoplasma pathogenesis, we generated Cfp4-deficient strains by insertional mutagenesis and by RNA interference. Cfp4-deficient strains are not attenuated in virulence in human macrophages nor during lung infection in a murine model of histoplasmosis. Co-infection of differentially marked Cfp4-producing and Cfp4-deficient strains demonstrates that production of Cfp4 does not confer a fitness advantage to Histoplasma yeasts during murine lung infection. Despite no apparent role in acute virulence in mice, secretion of the Cfp4 glycoprotein by yeast cells is consistent across clinical and laboratory isolates of the North American type 1 and type 2 phylogenetic groups as well a strain from Panama. In addition, human immune sera recognize the Histoplasma Cfp4 protein, confirming Cfp4 production during infection of human hosts. These results suggest the potential utility of Cfp4 as a diagnostic exoantigen for histoplasmosis.
