CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity

Blondel, Carlos J.; Park, Joseph S.; Hubbard, Troy P.; Pacheco, Alline R.; Kuehl, Carole J.; Walsh, Michael J.; Davis, Brigid M.; Gewurz, Benjamin E.; Doench, John G.; Waldor, Matthew K.

doi:10.1016/j.chom.2016.06.010

Cited by 60 publications

(65 citation statements)

References 49 publications

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“…As large-scale genetic screens to identify epithelial cell factors that mediate interactions with intracellular pathogens are lacking, we performed a multi-round, genome-wide CRISPR/Cas9 loss-of-function screen in the human colonic epithelial cell line HT29-Cas9 (Blondel et al, 2016), to identify IEC genes that confer resistance to Stm cytotoxicity (Figure 1A). 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.…”

Section: Resultsmentioning

confidence: 99%

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Functional remodeling of lysosomes by type I interferon modifies host defense

Zhang

Zoued

Liu

et al. 2020

Preprint

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SUMMARYOrganelle remodeling is critical for cellular homeostasis, but host factors that control organelle function during microbial infection remain largely uncharacterized. Here, a genome-scale CRISPR/Cas9 screen in intestinal epithelial cells with the prototypical intracellular bacterial pathogen Salmonella led us to discover that type I interferon (IFN-I) remodels lysosomes. Even in the absence of infection, IFN-I signaling modified the localization, acidification, protease activity and proteomic profile of lysosomes. Proteomic and genetic analyses revealed that multiple IFN-I-stimulated genes including Ifitm3, Slc15a3, and Cnp contribute to lysosome acidification. IFN-I-dependent lysosome acidification stimulated intracellular Salmonella virulence gene expression, leading to rupture of the Salmonella-containing vacuole and host cell death. Moreover, IFN-I signaling promoted in vivo Salmonella pathogenesis in the intestinal epithelium, where Salmonella initiates infection. Our findings explain how an intracellular bacterial pathogen co-opts epithelial IFN-I signaling. We propose that IFN-I control of lysosome function broadly impacts host defense against diverse viral and microbial pathogens.

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Section: Resultsmentioning

confidence: 99%

“…HT29-Cas9 CRISPR libraries were constructed as described previously (Blondel et al, 2016) using the Avana sgRNA library, which contains four different sgRNAs targeting each human protein-coding gene (Doench et al, 2016). For each library, two sets of four T225 flasks (Corning, Cat No.…”

Section: Methods Detailsmentioning

confidence: 99%

Functional remodeling of lysosomes by type I interferon modifies host defense

Zhang

Zoued

Liu

et al. 2020

Preprint

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“…HSPG synthesis and sulfation is driven by >20 different genes 28 ; as shown in Fig. 3c and d, the significant enrichment of specific sgRNAs identified 7 genes potentially involved in HSPG synthesis and metabolic pathways, and 2 genes potentially involved in sulfurylation modifications of HSPG 33,34 in porcine cells.…”

Section: Knockout Of Heparan Sulfate Proteoglycan (Hspg) Pathway Relamentioning

confidence: 85%

CRISPR screening of porcine sgRNA library identified host factors essential for Japanese encephalitis virus replication

Zhao

Liu

Xiao

et al. 2019

Preprint

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Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, key host genes involved in the JEV life cycle and cell death are poorly understood. Here, we designed 85,674 single guide RNAs (sgRNAs) targeting 17,743 protein-coding genes, 11,053 long ncRNAs (lncRNAs), and 551 microRNAs (miRNAs) in the porcine genome, and subsequently developed a porcine sgRNA library and genome-scale CRISPR/Cas9 knockout (PigGeCKO) system. These sgRNAs were delivered into porcine kidney-15 (PK-15) cells that constitutively express Cas9, positive selection screening of the resulting PigGeCKO cell collection for resistance to JEV-induced cell death led to the identification of several previously unreported genes required for JEV infection. We conducted follow-up studies to verify the dependency of JEV on these genes, and identified functional contributions for six of the many candidate JEV-related host genes, including an endoplasmic reticulum membrane protein complex subunit 3 (EMC3) and calreticulin (CALR). Additionally, we identified that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPG sulfurylation, facilitated JEV entry into PK-15 cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this work deepens our basic understanding of flavivirus infection and identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to prevent and treat diseases caused by Japanese encephalitis virus.

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“…RNA interference screens have been utilized to study mycobacterial infection (Kumar et al, 2010; Li et al, 2016; Philips et al, 2005). CRISPR-Cas9-mediated genome-wide screens, displaying high efficiency and minimal off-target effects (Shalem et al, 2014; Wang et al, 2014), revealed host cell responses to non-mycobacterial virulence factors (Blondel et al, 2016; Chang et al, 2019; Pacheco et al, 2018; Parnas et al, 2015; Tao et al, 2016), and a CRISPR interference (CRISPRi)-mediated repression library was used to elucidate the function of essential genes and long noncoding RNAs in host cells responding to cholera-diphtheria toxin (Gilbert et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

Lai

Babunovic

Cui

et al. 2020

Preprint

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222 SUMMARY: 23 Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death 24 worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating 25 the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated 26 high-throughput genetic screens to identify perturbations that improve the survival of 27 human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-28 Gué rin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations 29 constrained the growth of intracellular mycobacteria. We identified over 100 genes 30 associated with diverse biological pathways as potential HDT targets. We validated key 31 components of the type I interferon and aryl hydrocarbon receptor signaling pathways that 32 respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these 33 inhibitors enhanced human macrophage survival and limited the intracellular growth of 34 Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex host-35 pathogen interactions and serve to identify HDTs with the potential to improve TB 36 treatment. 37 38

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CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity

Cited by 60 publications

References 49 publications

Functional remodeling of lysosomes by type I interferon modifies host defense

Functional remodeling of lysosomes by type I interferon modifies host defense

CRISPR screening of porcine sgRNA library identified host factors essential for Japanese encephalitis virus replication

Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

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