High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>

ABSTRACT Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Marcin Dembek, Lars Barquist, Christine J. Boinett, Amy K. Cain, Matthew Mayho, Trevor D. Lawley, Neil F. Fairweather, Robert P. Fagan
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/e216797dfeeb4627b710e0978c35925a
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:e216797dfeeb4627b710e0978c35925a
record_format	dspace
spelling	oai:doaj.org-article:e216797dfeeb4627b710e0978c35925a2021-11-15T15:41:33ZHigh-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>10.1128/mBio.02383-142150-7511https://doaj.org/article/e216797dfeeb4627b710e0978c35925a2015-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02383-14https://doaj.org/toc/2150-7511ABSTRACT Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.Marcin DembekLars BarquistChristine J. BoinettAmy K. CainMatthew MayhoTrevor D. LawleyNeil F. FairweatherRobert P. FaganAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 2 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Marcin Dembek Lars Barquist Christine J. Boinett Amy K. Cain Matthew Mayho Trevor D. Lawley Neil F. Fairweather Robert P. Fagan High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
description	ABSTRACT Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen. IMPORTANCE Clostridium difficile is a common cause of potentially fatal intestinal infections in hospital patients, particularly those who have been treated with antibiotics. Our knowledge of this bacterium has been hampered by a lack of tools for dissecting the organism. We have developed a method to study the function of every gene in the bacterium simultaneously. Using this tool, we have identified a set of 404 genes that are required for growth of the bacteria in the laboratory. C. difficile also produces a highly resistant spore that can survive in the environment for a long time and is a requirement for transmission of the bacteria between patients. We have applied our genetic tool to identify all of the genes required for production of a spore. All of these genes represent attractive targets for new drugs to treat infection.
format	article
author	Marcin Dembek Lars Barquist Christine J. Boinett Amy K. Cain Matthew Mayho Trevor D. Lawley Neil F. Fairweather Robert P. Fagan
author_facet	Marcin Dembek Lars Barquist Christine J. Boinett Amy K. Cain Matthew Mayho Trevor D. Lawley Neil F. Fairweather Robert P. Fagan
author_sort	Marcin Dembek
title	High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
title_short	High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
title_full	High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
title_fullStr	High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
title_full_unstemmed	High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>
title_sort	high-throughput analysis of gene essentiality and sporulation in <named-content content-type="genus-species">clostridium difficile</named-content>
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/e216797dfeeb4627b710e0978c35925a
work_keys_str_mv	AT marcindembek highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT larsbarquist highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT christinejboinett highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT amykcain highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT matthewmayho highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT trevordlawley highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT neilffairweather highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent AT robertpfagan highthroughputanalysisofgeneessentialityandsporulationinnamedcontentcontenttypegenusspeciesclostridiumdifficilenamedcontent
_version_	1718427676564258816

High-Throughput Analysis of Gene Essentiality and Sporulation in <named-content content-type="genus-species">Clostridium difficile</named-content>

Ejemplares similares