Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death

Shahi, Ifrah; Llaneras, Cristina N.; Perelman, Sofya S.; Torres, Victor J.; Ratner, Adam J.

doi:10.1128/spectrum.02186-21

Cited by 6 publications

(9 citation statements)

References 46 publications

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“…We used cells that were transduced with CRISPR/Cas9 single guide RNA targeting the hCD59 gene [HeLa hCD59 knockout (KO)] and HeLa control cells that had been transduced with a non-targeting sgRNA (HeLa NT). We have previously used these cell lines to successfully examine ILY activity and hCD59-dependence [ 29 ]. The two recombinant TGY variants were tested for lysis on the HeLa NT cells and HeLa hCD59 KO cells, and IC 50 values (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…NT control and hCD59 KO cell lines were generated as previously described [ 29 ]. Briefly, single guide RNAs (sgRNAs) were ligated into an empty pLentiCRISPR plasmid backbone (gifted by Dr Feng Zhang; Addgene plasmid #52 961 [ 30 ]) and packaged into lentivirus using XtremeGENE 9 DNA Transfection Reagent.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were washed with ice-cold PBS, and resuspended in 300 µl of ice-cold PBS+3 % BSA+1 % sodium azide. Flow cytometry to visualize hCD59 expression was carried out using indirect staining as described previously [ 29 ]. Stained cells were visualized on a CytoFlex Analyzer flow cytometer, and the data were analysed using FlowJo software (version 10.8.1).…”

Section: Methodsmentioning

confidence: 99%

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Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity

Shahi,

Dongas,

Ilmain

et al. 2023

Microbiology

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Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp . tigurinus , tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp . tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity

Shahi,

Dongas,

Ilmain

et al. 2023

Microbiology

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“…In 2020, Drabavicius et al reported a CRISPR screen in near-haploid HAP1 cells for Streptococcus intermedius-produced CDC intermedilysin (ILY) [138], which has been known to utilize a GPI-anchored protein CD59 as a receptor [139,140]. This screen, along with a similar one in HeLa cells reported by Shahi et al [141], successfully identified the known ILY receptor CD59 and a whole bunch of host factors in the GPI biosynthesis pathway (e.g., PIGA and PIGB). Drabavicius et al also identified a panel of genes involved in various cellular processes.…”

Section: Other Toxin-based Screens For Glycosylation Pathwaysmentioning

confidence: 86%

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens

Tian

Zhou

2023

Bioengineering

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Genetic screen technology has been applied to study the mechanism of action of bacterial toxins—a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

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“…Except in the capsular polysaccharide genes, relA was identified to be essential for GBS survival in blood. In addition to promoting the persistence of GBS in host blood, the activation of a stringent response by relA can also enhance the expression of β-hemolysin/cytolysin, a toxin required for GBS virulence [ 58 ] through an arginine-mediated metabolism pathway. At present, a small molecule inhibitor aimed at the stringent response has been developed as a new type of antibacterial, and it has demonstrated the corresponding therapeutic effects in pre-clinical tests [ 59 , 60 ].…”

Section: Research On the Mechanism Of Bacterial Persistencementioning

confidence: 99%

Recent Advances in Bacterial Persistence Mechanisms

Pan,

Liu,

et al. 2023

IJMS

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The recurrence of bacterial infectious diseases is closely associated with bacterial persisters. This subpopulation of bacteria can escape antibiotic treatment by entering a metabolic status of low activity through various mechanisms, for example, biofilm, toxin–antitoxin modules, the stringent response, and the SOS response. Correspondingly, multiple new treatments are being developed. However, due to their spontaneous low abundance in populations and the lack of research on in vivo interactions between persisters and the host’s immune system, microfluidics, high-throughput sequencing, and microscopy techniques are combined innovatively to explore the mechanisms of persister formation and maintenance at the single-cell level. Here, we outline the main mechanisms of persister formation, and describe the cutting-edge technology for further research. Despite the significant progress regarding study techniques, some challenges remain to be tackled.

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Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae β-Hemolysin/Cytolysin-Induced Cell Death

Abstract: CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins.

Cited by 6 publications

References 46 publications

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens

Recent Advances in Bacterial Persistence Mechanisms

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