{"title":"The Pvc15 ATPase selectively associates effector proteins with the <i>Photorhabdus</i> virulence cassette.","authors":"Rhys Evans, Nicholas R Waterfield","doi":"10.1098/rsos.240948","DOIUrl":null,"url":null,"abstract":"<p><p>The <i>Photorhabdus</i> virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of <i>Escherichia coli</i> lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.</p>","PeriodicalId":21525,"journal":{"name":"Royal Society Open Science","volume":"11 10","pages":"240948"},"PeriodicalIF":2.9000,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11495950/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Royal Society Open Science","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.1098/rsos.240948","RegionNum":3,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.
期刊介绍:
Royal Society Open Science is a new open journal publishing high-quality original research across the entire range of science on the basis of objective peer-review.
The journal covers the entire range of science and mathematics and will allow the Society to publish all the high-quality work it receives without the usual restrictions on scope, length or impact.