A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection

Abstract

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13. IMPORTANCE Salmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Functional remodeling of lysosomes by type I interferon modifies host defense

“…HT29 cells are efficiently invaded and subsequently killed by Stm, providing a strong selective force to enrich for guide RNAs targeting host factors that modulate cytotoxicity. The screen identified known pathways that sensitize cells to Stm infection, including those involved in regulation of actin dynamics (Arp2/3 and Rac), which are important in pathogen invasion (Table S1 and Figures 1B, C), (Unsworth et al, 2004; Yeung et al, 2019). Genes linked to pathways not previously directly linked to Stm virulence, including the Fc-gamma receptor-dependent phagocytic and GPI anchor modification pathways were also enriched (Figures 1B, C).…”

Variant in NHLRC2 leads to increased hnRNP C2 in developing neurons and the hippocampus of a mouse model of FINCA disease

“…Dot blots show individual data points, group mean and standard error of the mean (SEM). ****p < 0.0001, ***p < 0.001 processes (Paakkola et al 2018;Haney et al 2018;Yeung et al 2019).…”

Utilizing Organoid and Air-Liquid Interface Models as a Screening Method in the Development of New Host Defense Peptides

“…Macrophages derived from human iPSCs established by Hale et al have been harnessed to generate both wild-type and mutant varieties (Hale et al, 2015;Yeung et al, 2017Yeung et al, , 2019. The generated iPSDMs were compared to blood monocyte derived macrophages and found to comparably phagocytize Chlamydia and generate very similar gene expression responses (Hale et al, 2015).…”

“…Yeung et al utilized a mutant library of Cas9-expressing THP-1 cells and screened these for their inability to take up Salmonella. She consequently identified 183 loci that are required for Salmonella invasion of macrophages, each of which represented a potential target for host-directed therapy (Yeung et al, 2019). Such a strategy is easily translatable to iPSDMs since iPSCs are very genetically malleable.…”