A remarkable expansion of secreted cysteine knot proteins reveals novel virulence factors in the human fungal pathogen <i>Histoplasma</i>

Identifying fungal secreted factors that influence host infection remains a key challenge in microbial pathogenesis. While secreted effectors, particularly small cysteine-rich proteins, are well characterized in plant fungal pathogens, their counterparts in mammalian pathogens remain elusive. We applied criteria from plant fungal effectors to the mammalian fungal pathogen <i>Histoplasma</i> , yielding a set of putative effectors highly enriched for knottins, proteins that adopt a distinctive cysteine knot fold. Using a novel algorithm, we further identified 25 putative knottins in the <i>Histoplasma</i> genome, revealing a significant expansion of knottin genes. Knottin domains are found in diverse molecules but play an unknown role in virulence. Functional studies of individual <i>Histoplasma</i> knottins demonstrated their critical roles in intracellular survival and host cell lysis during macrophage infection as well as virulence <i>in vivo</i> . These findings highlight the importance of knottins in fungal pathogenesis and suggest their broader relevance for discovering conserved mechanisms of host manipulation.