{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T03:15:10Z","timestamp":1792293310239,"version":"4.4.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1013021","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T00:00:00Z","timestamp":1744243200000}}],"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2025,4,2]],"date-time":"2025-04-02T00:00:00Z","timestamp":1743552000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32 HL134598"],"award-info":[{"award-number":["T32 HL134598"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30 DK117469"],"award-info":[{"award-number":["P30 DK117469"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000897","name":"Cystic Fibrosis Foundation","doi-asserted-by":"publisher","award":["STANTO19R0"],"award-info":[{"award-number":["STANTO19R0"]}],"id":[{"id":"10.13039\/100000897","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Philip Hanlon and Gail Gentes Cluster for Personalized Treatments for Cystic Fibrosis"}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p><jats:italic>Pseudomonas aeruginosa<\/jats:italic> is an opportunistic pathogen responsible for airway infections in immunocompromised individuals, including those with cystic fibrosis (CF). <jats:italic>P. aeruginosa<\/jats:italic> has a type III secretion system (T3SS) that translocates effectors into host cells. ExoS is a T3SS effector with ADP ribosyltransferase (ADPRT) activity. ExoS ADPRT activity promotes <jats:italic>P. aeruginosa<\/jats:italic> virulence by inhibiting phagocytosis and limiting oxidative burst in neutrophils. The <jats:italic>P. aeruginosa<\/jats:italic> T3SS also translocates flagellin, which can activate the NLRC4 inflammasome, resulting in: 1) gasdermin-D pores, release of IL-1\u03b2 and pyroptosis; and 2) histone 3 citrullination (CitH3), nuclear DNA decondensation and expansion into the neutrophil cytosol with incomplete NET extrusion. However, studies with <jats:italic>P. aeruginosa<\/jats:italic> PAO1 indicate that ExoS ADPRT activity inhibits the NLRC4 inflammasome in neutrophils. Here, we identified an ExoS<jats:sup>+<\/jats:sup> CF clinical isolate of <jats:italic>P. aeruginosa<\/jats:italic> with a hyperactive T3SS. Variants of the hyperactive T3SS mutant or PAO1 were used to infect neutrophils from C57BL\/6 mice that were wildtype or engineered to have a CF genotype or defects in inflammasome assembly. Responses to NLRC4 inflammasome assembly or ExoS ADPRT activity were assayed and found to be similar for C57BL\/6 or CF neutrophils. ExoS ADPRT activity in the hyperactive T3SS mutant regulated inflammasome, nuclear DNA decondensation and incomplete NET extrusion responses, like PAO1, but promoted enhanced CitH3 and plasma membrane rupture (PMR). Glycine supplementation inhibited PMR by the hyperactive T3SS mutant, suggesting ninjurin-1 is required for this process. These results identify enhanced neutrophil PMR as a pathogenic activity of ExoS ADPRT in hypervirulent <jats:italic>P. aeruginosa<\/jats:italic>.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013021","type":"journal-article","created":{"date-parts":[[2025,4,2]],"date-time":"2025-04-02T23:27:45Z","timestamp":1743636465000},"page":"e1013021","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":11,"title":["ExoS effector in Pseudomonas aeruginosa Hyperactive Type III secretion system mutant promotes enhanced Plasma Membrane Rupture in Neutrophils"],"prefix":"10.1371","volume":"21","author":[{"given":"Arianna D.","family":"Reuven","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Katzenell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bethany W.","family":"Mwaura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6047-8837","authenticated-orcid":true,"given":"James B.","family":"Bliska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,4,2]]},"reference":[{"issue":"1","key":"ppat.1013021.ref001","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1128\/CMR.00036-10","article-title":"Clinical significance of microbial infection and adaptation in cystic fibrosis","volume":"24","author":"AR Hauser","year":"2011","journal-title":"Clin Microbiol Rev"},{"issue":"3","key":"ppat.1013021.ref002","doi-asserted-by":"crossref","first-page":"e00138","DOI":"10.1128\/CMR.00138-18","article-title":"Cystic Fibrosis and Pseudomonas aeruginosa: the Host-Microbe Interface","volume":"32","author":"S Malhotra","year":"2019","journal-title":"Clin Microbiol Rev"},{"issue":"8","key":"ppat.1013021.ref003","doi-asserted-by":"crossref","first-page":"831","DOI":"10.2174\/138920312804871210","article-title":"Structure and function of the Type III secretion system of Pseudomonas aeruginosa","volume":"13","author":"M Galle","year":"2012","journal-title":"Curr Protein Pept Sci"},{"issue":"9","key":"ppat.1013021.ref004","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/nrmicro2199","article-title":"The type III secretion system of Pseudomonas aeruginosa: infection by injection","volume":"7","author":"AR Hauser","year":"2009","journal-title":"Nat Rev Microbiol"},{"issue":"1","key":"ppat.1013021.ref005","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1038\/s41467-022-28967-5","article-title":"CrkII\/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT","volume":"13","author":"MF Mohamed","year":"2022","journal-title":"Nat Commun"},{"issue":"Pt 10","key":"ppat.1013021.ref006","doi-asserted-by":"crossref","first-page":"2659","DOI":"10.1099\/00221287-147-10-2659","article-title":"Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa","volume":"147","author":"H Feltman","year":"2001","journal-title":"Microbiology (Reading)"},{"issue":"3","key":"ppat.1013021.ref007","doi-asserted-by":"crossref","first-page":"e01080-14","DOI":"10.1128\/mBio.01080-14","article-title":"The ADP-ribosyltransferase domain of the effector protein ExoS inhibits phagocytosis of Pseudomonas aeruginosa during pneumonia","volume":"5","author":"SM Rangel","year":"2014","journal-title":"mBio"},{"issue":"5","key":"ppat.1013021.ref008","doi-asserted-by":"crossref","first-page":"611-618.e5","DOI":"10.1016\/j.chom.2017.04.001","article-title":"Pseudomonas aeruginosa Effector ExoS Inhibits ROS Production in Human Neutrophils","volume":"21","author":"C Vareechon","year":"2017","journal-title":"Cell Host Microbe"},{"issue":"12","key":"ppat.1013021.ref009","doi-asserted-by":"crossref","first-page":"6969","DOI":"10.1128\/IAI.72.12.6969-6977.2004","article-title":"Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung","volume":"72","author":"CM Shaver","year":"2004","journal-title":"Infect Immun"},{"issue":"4","key":"ppat.1013021.ref010","doi-asserted-by":"crossref","first-page":"1884","DOI":"10.4049\/jimmunol.1102148","article-title":"ExoS and ExoT ADP ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival","volume":"188","author":"Y Sun","year":"2012","journal-title":"J Immunol"},{"issue":"5","key":"ppat.1013021.ref011","doi-asserted-by":"crossref","first-page":"961","DOI":"10.1111\/mmi.14891","article-title":"Inflammasome and gasdermin signaling in neutrophils","volume":"117","author":"SJ Yow","year":"2022","journal-title":"Mol Microbiol"},{"issue":"3","key":"ppat.1013021.ref012","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1038\/mi.2016.73","article-title":"Neutrophil pyroptosis mediates pathology of P. aeruginosa lung infection in the absence of the NADPH oxidase NOX2","volume":"10","author":"J-C Ryu","year":"2017","journal-title":"Mucosal Immunol"},{"issue":"7","key":"ppat.1013021.ref013","doi-asserted-by":"crossref","first-page":"3097","DOI":"10.4049\/jimmunol.1501642","article-title":"Clinical Isolates of Pseudomonas aeruginosa from Chronically Infected Cystic Fibrosis Patients Fail To Activate the Inflammasome during Both Stable Infection and Pulmonary Exacerbation","volume":"196","author":"KE Huus","year":"2016","journal-title":"J Immunol"},{"issue":"8","key":"ppat.1013021.ref014","doi-asserted-by":"crossref","first-page":"111688","DOI":"10.1016\/j.celrep.2022.111688","article-title":"Neutrophil inflammasomes sense the subcellular delivery route of translocated bacterial effectors and toxins","volume":"41","author":"C Oh","year":"2022","journal-title":"Cell Rep"},{"issue":"7","key":"ppat.1013021.ref015","doi-asserted-by":"crossref","first-page":"e1010305","DOI":"10.1371\/journal.ppat.1010305","article-title":"Caspase-1-driven neutrophil pyroptosis and its role in host susceptibility to Pseudomonas aeruginosa","volume":"18","author":"K Santoni","year":"2022","journal-title":"PLoS Pathog"},{"issue":"1","key":"ppat.1013021.ref016","doi-asserted-by":"crossref","first-page":"5832","DOI":"10.1038\/s41467-023-41391-7","article-title":"NLRP3 selectively drives IL-1\u03b2 secretion by Pseudomonas aeruginosa infected neutrophils and regulates corneal disease severity","volume":"14","author":"MS Minns","year":"2023","journal-title":"Nat Commun"},{"key":"ppat.1013021.ref017","doi-asserted-by":"crossref","first-page":"1670","DOI":"10.3389\/fimmu.2019.01670","article-title":"Regulation of Pseudomonas aeruginosa-Mediated Neutrophil Extracellular Traps","volume":"10","author":"S Skopelja-Gardner","year":"2019","journal-title":"Front Immunol"},{"key":"ppat.1013021.ref018","doi-asserted-by":"crossref","first-page":"15010","DOI":"10.1038\/nrdp.2015.10","article-title":"Cystic fibrosis","volume":"1","author":"F Ratjen","year":"2015","journal-title":"Nat Rev Dis Primers"},{"issue":"1","key":"ppat.1013021.ref019","doi-asserted-by":"crossref","first-page":"1780","DOI":"10.1093\/gbe\/evz119","article-title":"The Population Structure of Pseudomonas aeruginosa Is Characterized by Genetic Isolation of exoU+ and exoS+ Lineages","volume":"11","author":"EA Ozer","year":"2019","journal-title":"Genome Biol Evol"},{"issue":"7","key":"ppat.1013021.ref020","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1164\/rccm.201409-1646OC","article-title":"Divergent, coexisting Pseudomonas aeruginosa lineages in chronic cystic fibrosis lung infections","volume":"191","author":"D Williams","year":"2015","journal-title":"Am J Respir Crit Care Med"},{"issue":"6","key":"ppat.1013021.ref021","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/j.trsl.2008.10.003","article-title":"Evolution of Pseudomonas aeruginosa type III secretion in cystic fibrosis: a paradigm of chronic infection","volume":"152","author":"M Jain","year":"2008","journal-title":"Transl Res"},{"issue":"2","key":"ppat.1013021.ref022","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1086\/338197","article-title":"Children with cystic fibrosis produce an immune response against exoenzyme S, a type III cytotoxin of Pseudomonas aeruginosa","volume":"185","author":"B Banwart","year":"2002","journal-title":"J Infect Dis"},{"issue":"5","key":"ppat.1013021.ref023","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1164\/rccm.202007-2735LE","article-title":"Cystic Fibrosis Lung Function Decline after Within-Host Evolution Increases Virulence of Infecting Pseudomonas aeruginosa","volume":"203","author":"P Jorth","year":"2021","journal-title":"Am J Respir Crit Care Med"},{"issue":"3","key":"ppat.1013021.ref024","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/j.chom.2015.07.006","article-title":"Regional Isolation Drives Bacterial Diversification within Cystic Fibrosis Lungs","volume":"18","author":"P Jorth","year":"2015","journal-title":"Cell Host Microbe"},{"issue":"12","key":"ppat.1013021.ref025","doi-asserted-by":"crossref","first-page":"3811","DOI":"10.1128\/JB.00002-09","article-title":"Functional domains of ExsA, the transcriptional activator of the Pseudomonas aeruginosa type III secretion system","volume":"191","author":"ED Brutinel","year":"2009","journal-title":"J Bacteriol"},{"key":"ppat.1013021.ref026","doi-asserted-by":"crossref","first-page":"e78609","DOI":"10.7554\/eLife.78609","article-title":"Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death","volume":"11","author":"JP Borges","year":"2022","journal-title":"Elife"},{"issue":"6","key":"ppat.1013021.ref027","doi-asserted-by":"crossref","first-page":"886","DOI":"10.1189\/jlb.1104636","article-title":"The expression and roles of Toll-like receptors in the biology of the human neutrophil","volume":"77","author":"LC Parker","year":"2005","journal-title":"J Leukoc Biol"},{"key":"ppat.1013021.ref028","doi-asserted-by":"crossref","first-page":"217","DOI":"10.3389\/fcimb.2017.00217","article-title":"Multiple Phenotypic Changes Define Neutrophil Priming","volume":"7","author":"I Miralda","year":"2017","journal-title":"Front Cell Infect Microbiol"},{"issue":"3","key":"ppat.1013021.ref029","doi-asserted-by":"crossref","first-page":"109829","DOI":"10.1016\/j.celrep.2021.109829","article-title":"Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis","volume":"37","author":"CR Armbruster","year":"2021","journal-title":"Cell Rep"},{"issue":"3","key":"ppat.1013021.ref030","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.prrv.2008.04.003","article-title":"Nasal and sinus disease in cystic fibrosis","volume":"9","author":"JM Robertson","year":"2008","journal-title":"Paediatr Respir Rev"},{"issue":"6","key":"ppat.1013021.ref031","doi-asserted-by":"crossref","first-page":"1479","DOI":"10.1128\/JB.01457-09","article-title":"ExsD inhibits expression of the Pseudomonas aeruginosa type III secretion system by disrupting ExsA self-association and DNA binding activity","volume":"192","author":"ED Brutinel","year":"2010","journal-title":"J Bacteriol"},{"issue":"1","key":"ppat.1013021.ref032","doi-asserted-by":"crossref","first-page":"9881","DOI":"10.1038\/s41598-020-66555-z","article-title":"Backbone Interactions Between Transcriptional Activator ExsA and Anti-Activator ExsD Facilitate Regulation of the Type III Secretion System in Pseudomonas aeruginosa","volume":"10","author":"M Shrestha","year":"2020","journal-title":"Sci Rep"},{"issue":"8","key":"ppat.1013021.ref033","doi-asserted-by":"crossref","first-page":"e0136533","DOI":"10.1371\/journal.pone.0136533","article-title":"Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa","volume":"10","author":"M Shrestha","year":"2015","journal-title":"PLoS One"},{"issue":"8","key":"ppat.1013021.ref034","doi-asserted-by":"crossref","first-page":"L902","DOI":"10.1152\/ajplung.00228.2015","article-title":"Differential ASC requirements reveal a key role for neutrophils and a noncanonical IL-1\u03b2 response to Pseudomonas aeruginosa","volume":"309","author":"YR Patankar","year":"2015","journal-title":"Am J Physiol Lung Cell Mol Physiol"},{"issue":"7967","key":"ppat.1013021.ref035","doi-asserted-by":"crossref","first-page":"1065","DOI":"10.1038\/s41586-023-05991-z","article-title":"Structural basis of NINJ1-mediated plasma membrane rupture in cell death","volume":"618","author":"M Degen","year":"2023","journal-title":"Nature"},{"issue":"7848","key":"ppat.1013021.ref036","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/s41586-021-03218-7","article-title":"NINJ1 mediates plasma membrane rupture during lytic cell death","volume":"591","author":"N Kayagaki","year":"2021","journal-title":"Nature"},{"key":"ppat.1013021.ref037","doi-asserted-by":"crossref","first-page":"2531","DOI":"10.1099\/00221287-146-10-2531","article-title":"Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of exoS","author":"MR Kaufman","year":"2000","journal-title":"Microbiology (Reading)"},{"issue":"7","key":"ppat.1013021.ref038","doi-asserted-by":"crossref","first-page":"2016","DOI":"10.4049\/jimmunol.1800076","article-title":"Dysregulated Calcium Homeostasis in Cystic Fibrosis Neutrophils Leads to Deficient Antimicrobial Responses","volume":"201","author":"FH Robledo-Avila","year":"2018","journal-title":"J Immunol"},{"issue":"4","key":"ppat.1013021.ref039","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1046\/j.1365-2958.1998.01106.x","article-title":"Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells","volume":"30","author":"KJ Pederson","year":"1998","journal-title":"Mol Microbiol"},{"key":"ppat.1013021.ref040","doi-asserted-by":"crossref","first-page":"e76555","DOI":"10.7554\/eLife.76555","article-title":"Metabolic basis for the evolution of a common pathogenic Pseudomonas aeruginosa variant","volume":"11","author":"DL Mould","year":"2022","journal-title":"Elife"},{"issue":"5","key":"ppat.1013021.ref041","doi-asserted-by":"crossref","first-page":"e0209821","DOI":"10.1128\/mBio.02098-21","article-title":"The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection","volume":"12","author":"NA Loeven","year":"2021","journal-title":"mBio"},{"issue":"3","key":"ppat.1013021.ref042","doi-asserted-by":"crossref","first-page":"1900","DOI":"10.4049\/jimmunol.0900612","article-title":"Selective expansion of the monocytic lineage directed by bacterial infection","volume":"183","author":"NV Serbina","year":"2009","journal-title":"J Immunol"},{"issue":"6996","key":"ppat.1013021.ref043","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1038\/nature02664","article-title":"Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf","volume":"430","author":"S Mariathasan","year":"2004","journal-title":"Nature"},{"issue":"6","key":"ppat.1013021.ref044","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: making protein folding accessible to all","volume":"19","author":"M Mirdita","year":"2022","journal-title":"Nat Methods"},{"issue":"4","key":"ppat.1013021.ref045","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.ymeth.2012.07.018","article-title":"The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli","volume":"58","author":"A Battesti","year":"2012","journal-title":"Methods"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1013021","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T00:00:00Z","timestamp":1744243200000}},{"DOI":"10.1371\/journal.ppat.1014353","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T00:00:00Z","timestamp":1782086400000}}],"container-title":["PLOS Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1013021","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T17:38:39Z","timestamp":1744306719000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.ppat.1013021"}},"subtitle":[],"editor":[{"given":"Vincent T","family":"Lee","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,4,2]]},"references-count":45,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4,2]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.ppat.1013021","relation":{},"ISSN":["1553-7374"],"issn-type":[{"value":"1553-7374","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,2]]}}}