Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

Carette, Jan E.; Guimarães, Carla P.; Varadarajan, Malini; Park, Annie S.; Wuethrich, Irene; Godarova, Alzbeta; Kotecki, Maciej; Cochran, Brent; Spooner, Eric; Ploegh, Hidde L.; Brummelkamp, Thijn R.

doi:10.1126/science.1178955

Cited by 459 publications

(595 citation statements)

References 33 publications

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“…To discover host factors required for α-toxin cytotoxicity, we conducted a live/dead genetic screen by intoxicating haploid human cells (HAP1) carrying knockout alleles in essentially all genes through insertional mutagenesis (22,23). A large-scale library of ∼100 million HAP1 mutagenized cells was treated with recombinant α-toxin, and gene-trap insertion sites were identified in the pool of surviving, toxin-resistant cells.…”

Section: Resultsmentioning

confidence: 99%

“…HAP1 cells were mutagenized with a retroviral gene trap to cause inactivating mutations throughout the genome, and a haploid genetic screen was performed as previously described (22,23). For a complete description of the haploid genetic screen, see SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…To advance understanding of how S. aureus α-toxin modulates host cell biology, we conducted a high-throughput genetic screen using human cells (22,23) to discover novel host factors required Significance Staphylococcus aureus is a major cause of invasive bacterial infection. One prominent virulence factor is α-toxin, a protein that injures the cell by forming a damaging pore across the cell membrane.…”

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confidence: 99%

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The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

Popov

Marceau

Starkl

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7 −/− mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.Staphylococcus aureus | α-toxin | adherens junctions | MRSA | PLEKHA7

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

Popov

Marceau

Starkl

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Haploid genetics has long provided an invaluable tool for basic genetic research in both prokaryote and eukaryote microbial systems [17][18][19] and has recently been extended to mammalian systems through the generation of haploid cell lines 20,21 . However, although Arabidopsis remains a premiere model system for basic research in higher plants, haploid genetics has been largely inaccessible to the Arabidopsis research community.…”

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A haploid genetics toolbox for Arabidopsis thaliana

et al. 2014

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Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.

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“…FP59 is a fusion protein of LF amino acids 1-254 and the catalytic domain of P. aeruginosa exotoxin A (15) that kills cells by ADP ribosylation of eEF2 after delivery to cytosol by PA. Using the toxin-resistant mutant cells combined with a genetic complementation or gene-trapping approaches, many proteins required for diphthamide biosynthesis have been identified in eukaryotes from yeast to humans, including OVCA1 (ovarian cancer 1, identical to Dph1), Dph2, Dph3, Dph4, Dph5, and WDR85 (YBR246W in yeast) (16,17). The biosynthesis of diphthamide represents one of the most complex posttranslational modifications, attesting to the importance of the diphthamide modification in eEF2 normal physiology (18).…”

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Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Liu

Bachran

Gupta

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly 717 Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly 717 -to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1 −/− mice, eEF2 G717R/G717R MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2 G717R/G717R mice showed milder phenotypes than OVCA1 −/− mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2 G717R/G717R /OVCA1 −/− double mutant mice displayed the milder phenotypes of the eEF2 G717R/G717R mice, suggesting that the embryonic lethality of OVCA1 −/− mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent −1 frameshifting during translation and show that the Gly 717 -to-Arg mutation cannot rescue this defect. E ukaryotic elongation factor 2 (eEF2) is a member of the GTPbinding translation elongation factor family, and an essential factor for protein synthesis and cell survival. eEF2 drives the GTP-dependent translocation of the nascent polypeptide chain from the A site to the P site of the ribosome and advances mRNA by three bases during the elongation cycle of protein synthesis (1). eEF2 is highly homologous in all eukaryotes. In fact, eEF2 of humans, rats, mice, hamsters, and other mammals have exactly the same amino acid sequence. Intriguingly, all eukaryotic eEF2 proteins contain a unique posttranslationally modified histidine residue termed diphthamide (2, 3). Diphthamide modification occurs after eEF2 is translated and is irreversible, marking the completion of the biosynthesis of eEF2.Although the physiological role of the diphthamide modification on eEF2 remains elusive, diphthamide is the well-known target for the adenosine diphosphate (ADP)-ribosylating toxins from bacterial pathogens, such as diphtheria toxin (DT) from Corynebacterium diphtheriae, Pseudomonas exotoxin A (ETA) from Pseudomonas aeruginosa, and the recently identified cholix toxin (CT) from Vibrio cholerae (4). As virulence factors, these ADP-ribosylating toxins catalyze transfer of the ADP ribose from nicotinamide adenine dinucleotide (NAD + ) to diphthamide on eEF2 (Fig. S1), thus inactivating eEF2, halting cellular protein synthesis, and causing cell death.Because the diphthamide modification is required for the action of the ADP-ribosylating toxins, the complex diphthamide biosynthesis pathway is amenable to genetic analysis, and mutants defective in diphthamide biosynthesis have been isolated in both Chinese hamster ovary (CHO) cells and yeast (Saccharomyces cerevisiae) by selection for resistance to DT or an engineered ADP-ribosylating toxin − anthrax protective a...

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Haploid Genetic Screens in Human Cells Identify Host Factors Used by Pathogens

Cited by 459 publications

References 33 publications

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

A haploid genetics toolbox for Arabidopsis thaliana

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

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