Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>

ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pat...

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Autores principales:	Irina Afonina, June Ong, Jerome Chua, Timothy Lu, Kimberly A. Kline
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Enterococcus faecalis CRISPR interference biofilms gene essentiality Ebp pili Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d07182183b6446c5907c0b2e9286b704
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spelling	oai:doaj.org-article:d07182183b6446c5907c0b2e9286b7042021-11-15T16:19:08ZMultiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>10.1128/mBio.01101-202150-7511https://doaj.org/article/d07182183b6446c5907c0b2e9286b7042020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01101-20https://doaj.org/toc/2150-7511ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.Irina AfoninaJune OngJerome ChuaTimothy LuKimberly A. KlineAmerican Society for MicrobiologyarticleEnterococcus faecalisCRISPR interferencebiofilmsgene essentialityEbp piliMicrobiologyQR1-502ENmBio, Vol 11, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Enterococcus faecalis CRISPR interference biofilms gene essentiality Ebp pili Microbiology QR1-502
spellingShingle	Enterococcus faecalis CRISPR interference biofilms gene essentiality Ebp pili Microbiology QR1-502 Irina Afonina June Ong Jerome Chua Timothy Lu Kimberly A. Kline Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
description	ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.
format	article
author	Irina Afonina June Ong Jerome Chua Timothy Lu Kimberly A. Kline
author_facet	Irina Afonina June Ong Jerome Chua Timothy Lu Kimberly A. Kline
author_sort	Irina Afonina
title	Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_short	Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_full	Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_fullStr	Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_full_unstemmed	Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>
title_sort	multiplex crispri system enables the study of stage-specific biofilm genetic requirements in <named-content content-type="genus-species">enterococcus faecalis</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/d07182183b6446c5907c0b2e9286b704
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Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in <named-content content-type="genus-species">Enterococcus faecalis</named-content>

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