UCSF

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. In chapter 2, to interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement C3 cleavage product, anaphylatoxin C3a. Although it is known that the serum complement system reacts with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage susceptibility to fungi has not been elucidated. In chapter 3, we demonstrated that, whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We explored the role of serum complement in macrophage phagocytosis of Hc, and found that the ability of serum to stimulate macrophage phagocytosis of Hc is dependent on complement, and involves C3a and C5a signaling, which can act redundantly to stimulate phagocytosis. We showed that C3aR localizes to the early phagosome during Hc infection, and coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also found that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. In chapter 4, we investigated the role of C3aR in the macrophage cytokine response to Hc and mouse susceptibility to Hc infection. Taken together, our results provide insight into host processes that affect Hc-macrophage interactions, and uncover a novel role for C3aR in macrophage recognition of fungi.