Sequencing of knottin deletion mutants of Histoplasma

Interrogation of Histoplasma, a human fungal pathogen of macrophages, provides an opportunity to discover fungal secreted factors that modulate pathogenesis in mammals. A plethora of effectors have been discovered in plant fungal pathogens, including small, secreted, cysteine-rich, proteins that are highly expressed during infection. These criteria were applied to identify putative Histoplasma effectors, which were highly enriched for proteins with homology to knottins, a cysteine knot gene family. We developed a novel algorithm that identified 25 putative knottins within the Histoplasma genome, indicative of a significant expansion of putative knottin genes in all Histoplasma species that was not observed across the fungal kingdom. Furthermore, characterization of four Histoplasma knottins revealed each was required for optimal growth within macrophages and/or host cell lysis. Two knottins were examined in vivo, uncovering a requirement for these effectors in virulence. Thus, our computational analysis is a discovery tool to identify novel virulence factors in a human fungal pathogen.