UCSF

Intracellular pathogens have developed strategies like secreting virulence factors to aid in replication and survival. Histoplasma capsulatum (Hc) is a thermally dimorphic human fungal pathogen that causes apoptosis of macrophages through the induction of the Integrated Stress Response (ISR). The mechanisms through which Hc induces host cell death are still poorly understood. In this work, we take a bioinformatic and genetic approach to identify and study novel Hc effectors. Known fungal effectors tend to be highly expressed in the pathogenic form of the organism, small, secreted, and cysteine-rich. Using these criteria, we identified many putative Hc effectors, some of which contained homology to the cysteine knot gene family called knottins. Knottins contain a 6-cysteine motif that creates three disulfide bridges forming a stable knot structure. We wrote a naïve algorithm to mine the Hc genome for knottins and identified a total of 26 putative knottins. Furthermore, we observed a unique and massive expansion of putative knottin genes in all Histoplasma species that was not observed across the fungal kingdom. We characterized four knottins named KNOT1, KNOT2, KNOT3 and KNOT4 and discovered that all four proteins localize to the host cytosol during Hc infection of macrophages. Using CRISPR-Cas9, we generated deletion mutants and discovered that KNOT1, KNOT2 and KNOT4 are dispensable for in vitro growth. Surprisingly, deletion of KNOT3 resulted in a moderate growth defect. We observed that KNOT1, KNOT3, and KNOT4 were all required for optimal Hc intracellular growth within macrophages as well as macrophage lysis. In contrast, KNOT2 was required for macrophage lysis but was dispensable for intracellular growth. Intriguingly, these knottins proteins were not required to stimulate a host pathway, the Integrated Stress Response (ISR), which was previously shown to be required for optimal macrophage lysis. Instead, we observe no difference in ISR induction in the absence of these knottins, suggesting that they interact with other host cellular pathways that are required for lysis. Of these four knottins, we chose to characterize KNOT2 and KNOT4 in the mouse model of infection. Mice failed to succumb to infection with mutant Hc strains lacking KNOT2 or KNOT4. Interestingly, the knot4Δ mutant displays a modest decrease in fungal burden in the mouse, whereas the knot2Δ mutant is able to grow to wild-type levels. These data indicate that KNOT2 and KNOT4 are dispensable for fungal burden and may instead manipulate other host processes such as the immune response. Taken together, this work identifies a new family of virulence factors and highlights how Hc is using a varied arsenal of effectors to cause disease.