阴道Sneathia细胞致病毒素A基因的靶向缺失

IF 2.6 2区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Rishi M. Ray, Phoebe V. Bridy, Aubree G. Musicant, Shiny Chandravel, Youstina Y. Aziz, Jasmine C. Cruz, Kimberly K. Jefferson
{"title":"阴道Sneathia细胞致病毒素A基因的靶向缺失","authors":"Rishi M. Ray, Phoebe V. Bridy, Aubree G. Musicant, Shiny Chandravel, Youstina Y. Aziz, Jasmine C. Cruz, Kimberly K. Jefferson","doi":"10.1111/mmi.70024","DOIUrl":null,"url":null,"abstract":"<jats:italic>Sneathia vaginalis</jats:italic> is a common component of the vaginal microbiome and is emerging as a marker for preterm birth. It produces the cytopathogenic toxin A (CptA), which is capable of lysing human red blood cells and permeabilizing epithelial cells. However, the role of CptA and other potential virulence factors in pathogenesis has been difficult to characterize due to the lack of genetic tools for targeted deletion in <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. The objective of this study was to create the first isogenic gene deletion mutant in <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. We chose the <jats:italic>cptA</jats:italic> gene as a target for deletion because of its role in virulence. We characterized the restriction‐modification profile in <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> to increase the chances that exogenous DNA would resist restriction digestion, and we identified an antibiotic resistance cassette that is functional in this species. We identified a genetic locus encoding a Dam methylase and a restriction endonuclease with DpnII‐like activity in <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> strain SN35. By convention, this newly described restriction endonuclease would be named SvaSI for <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> SN35. Using plasmid DNA purified from a Dam+ <jats:styled-content style=\"fixed-case\"><jats:italic>E. coli</jats:italic></jats:styled-content> strain to evade SvaSI restriction, we successfully replaced <jats:italic>cptA</jats:italic> with an erythromycin resistance cassette encoding the <jats:italic>ermF</jats:italic> and <jats:italic>ermAM</jats:italic> genes, creating the first genetically engineered deletion mutation in this species. Results revealed that CptA is necessary for the hemolytic and cytopathogenic activities of <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. This work is a resource that lays the foundation for the development of additional genetic tools for <jats:styled-content style=\"fixed-case\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> and facilitates the characterization of additional genes in this emerging pathogen.","PeriodicalId":19006,"journal":{"name":"Molecular Microbiology","volume":"25 1","pages":""},"PeriodicalIF":2.6000,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Targeted Deletion of the Cytopathogenic Toxin A Gene in Sneathia vaginalis\",\"authors\":\"Rishi M. Ray, Phoebe V. Bridy, Aubree G. Musicant, Shiny Chandravel, Youstina Y. Aziz, Jasmine C. Cruz, Kimberly K. Jefferson\",\"doi\":\"10.1111/mmi.70024\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<jats:italic>Sneathia vaginalis</jats:italic> is a common component of the vaginal microbiome and is emerging as a marker for preterm birth. It produces the cytopathogenic toxin A (CptA), which is capable of lysing human red blood cells and permeabilizing epithelial cells. However, the role of CptA and other potential virulence factors in pathogenesis has been difficult to characterize due to the lack of genetic tools for targeted deletion in <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. The objective of this study was to create the first isogenic gene deletion mutant in <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. We chose the <jats:italic>cptA</jats:italic> gene as a target for deletion because of its role in virulence. We characterized the restriction‐modification profile in <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> to increase the chances that exogenous DNA would resist restriction digestion, and we identified an antibiotic resistance cassette that is functional in this species. We identified a genetic locus encoding a Dam methylase and a restriction endonuclease with DpnII‐like activity in <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> strain SN35. By convention, this newly described restriction endonuclease would be named SvaSI for <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> SN35. Using plasmid DNA purified from a Dam+ <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>E. coli</jats:italic></jats:styled-content> strain to evade SvaSI restriction, we successfully replaced <jats:italic>cptA</jats:italic> with an erythromycin resistance cassette encoding the <jats:italic>ermF</jats:italic> and <jats:italic>ermAM</jats:italic> genes, creating the first genetically engineered deletion mutation in this species. Results revealed that CptA is necessary for the hemolytic and cytopathogenic activities of <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content>. This work is a resource that lays the foundation for the development of additional genetic tools for <jats:styled-content style=\\\"fixed-case\\\"><jats:italic>S. vaginalis</jats:italic></jats:styled-content> and facilitates the characterization of additional genes in this emerging pathogen.\",\"PeriodicalId\":19006,\"journal\":{\"name\":\"Molecular Microbiology\",\"volume\":\"25 1\",\"pages\":\"\"},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2025-09-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular Microbiology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1111/mmi.70024\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Microbiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1111/mmi.70024","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

阴道Sneathia是阴道微生物组的常见组成部分,并且正在成为早产的标志。它产生细胞致病性毒素A (CptA),能够裂解人红细胞和通透性上皮细胞。然而,由于缺乏靶向删除阴道链球菌的遗传工具,CptA和其他潜在毒力因子在发病机制中的作用一直难以表征。本研究的目的是在阴道链球菌中创建第一个等基因缺失突变体。我们选择cptA基因作为删除的目标,因为它在毒力中的作用。我们对阴道链球菌的限制性修饰谱进行了表征,以增加外源DNA抵抗限制性消化的机会,并确定了一个在该物种中起作用的抗生素耐药盒。我们在阴道链球菌SN35中发现了一个基因位点,编码一个具有DpnII -样活性的Dam甲基化酶和一个限制性内切酶。按照惯例,这种新描述的限制性内切酶将被命名为SvaSI,用于阴道链球菌SN35。利用从一株坝+大肠杆菌菌株中纯化的质粒DNA来逃避SvaSI限制,我们成功地用编码ermF和ermAM基因的红霉素耐药盒取代了cptA,在该物种中创造了第一个基因工程缺失突变。结果表明,CptA对阴道链球菌的溶血和细胞病变活性是必需的。这项工作为开发阴道链球菌的其他遗传工具奠定了基础,并促进了这种新兴病原体的其他基因的表征。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Targeted Deletion of the Cytopathogenic Toxin A Gene in Sneathia vaginalis
Sneathia vaginalis is a common component of the vaginal microbiome and is emerging as a marker for preterm birth. It produces the cytopathogenic toxin A (CptA), which is capable of lysing human red blood cells and permeabilizing epithelial cells. However, the role of CptA and other potential virulence factors in pathogenesis has been difficult to characterize due to the lack of genetic tools for targeted deletion in S. vaginalis. The objective of this study was to create the first isogenic gene deletion mutant in S. vaginalis. We chose the cptA gene as a target for deletion because of its role in virulence. We characterized the restriction‐modification profile in S. vaginalis to increase the chances that exogenous DNA would resist restriction digestion, and we identified an antibiotic resistance cassette that is functional in this species. We identified a genetic locus encoding a Dam methylase and a restriction endonuclease with DpnII‐like activity in S. vaginalis strain SN35. By convention, this newly described restriction endonuclease would be named SvaSI for S. vaginalis SN35. Using plasmid DNA purified from a Dam+ E. coli strain to evade SvaSI restriction, we successfully replaced cptA with an erythromycin resistance cassette encoding the ermF and ermAM genes, creating the first genetically engineered deletion mutation in this species. Results revealed that CptA is necessary for the hemolytic and cytopathogenic activities of S. vaginalis. This work is a resource that lays the foundation for the development of additional genetic tools for S. vaginalis and facilitates the characterization of additional genes in this emerging pathogen.
求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Molecular Microbiology
Molecular Microbiology 生物-生化与分子生物学
CiteScore
7.20
自引率
5.60%
发文量
132
审稿时长
1.7 months
期刊介绍: Molecular Microbiology, the leading primary journal in the microbial sciences, publishes molecular studies of Bacteria, Archaea, eukaryotic microorganisms, and their viruses. Research papers should lead to a deeper understanding of the molecular principles underlying basic physiological processes or mechanisms. Appropriate topics include gene expression and regulation, pathogenicity and virulence, physiology and metabolism, synthesis of macromolecules (proteins, nucleic acids, lipids, polysaccharides, etc), cell biology and subcellular organization, membrane biogenesis and function, traffic and transport, cell-cell communication and signalling pathways, evolution and gene transfer. Articles focused on host responses (cellular or immunological) to pathogens or on microbial ecology should be directed to our sister journals Cellular Microbiology and Environmental Microbiology, respectively.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:604180095
Book学术官方微信