Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

Abstract

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. 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 fragment C3a. Although it is known that complement components react with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage interactions with fungi has not been elucidated. 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 showed that C3aR localizes to the early phagosome during Hc infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells

“…In this manner, we identified Gβ subunits that either positively or negatively regulated phagocytosis. Consistent with prior work (38,39), HL-60 neutrophils lacking Gβ 2 exhibited a ~50% reduction in particle uptake (Fig. 1B).…”

“…Heterotrimeric G proteins regulate phagocytosis and migration in multiple myeloid cell types by transducing signals from seventransmembrane G protein-coupled receptors (GPCRs) (35)(36)(37)(38)(39)(40)(41)(42). GPCR activation induces dissociation of the G protein α subunit (Gα) from the βγ subcomplex (Gβγ), freeing both components to bind and activate specific downstream effector molecules (43).…”

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells

“…In this manner, we were able to identify G-subunits that either positively or negatively regulated phagocytosis. Consistent with prior work (Cohen et al 2022;Kamber et al 2021), HL-60 cells lacking G-2 exhibited a 50 percent reduction in particle uptake (Fig. 1b).…”

“…To identify novel mechanisms controlling phagocytosis and functional crosstalk, we performed a candidate screen focused on heterotrimeric G-protein signaling, which regulates phagocytosis and migration in multiple myeloid cell types(Peracino et al 1998; Wen et al 2019; Pan et al 2016; Cohen et al 2022; Kamber et al 2021; Yamada and Sixt 2019; Schwarz et al 2017; Sarris and Sixt 2015). Activating signals induce the dissociation of the G-protein α subunit (G α ) from the βγ subcomplex (G βγ ), freeing both components to bind and activate specific downstream effector molecules(Clapham and Neer 1997).…”

High-throughput CRISPR technology: a novel horizon for solid organ transplantation

“…In 2022, Sil et al. performed a host-directed CRISPR/Cas9 screen in the mouse mononuclear macrophage cell J774A.1 cell line to explore the interaction mechanism between H. capsulatum and macrophages ( 135 ). They found that the C3a receptor (C3aR) was important in macrophages capturing pathogenic fungi.…”