<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials

ABSTRACT Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gu...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Kurt Selle, Joshua R. Fletcher, Hannah Tuson, Daniel S. Schmitt, Lana McMillan, Gowrinarayani S. Vridhambal, Alissa J. Rivera, Stephanie A. Montgomery, Louis-Charles Fortier, Rodolphe Barrangou, Casey M. Theriot, David G. Ousterout
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Clostridioides difficile CRISPR-Cas Cas3 phage lysogeny CRISPR Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/05d3dd5ddad5489e8af88e62d8ba3612
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:05d3dd5ddad5489e8af88e62d8ba3612
record_format	dspace
spelling	oai:doaj.org-article:05d3dd5ddad5489e8af88e62d8ba36122021-11-15T15:57:01Z<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials10.1128/mBio.00019-202150-7511https://doaj.org/article/05d3dd5ddad5489e8af88e62d8ba36122020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00019-20https://doaj.org/toc/2150-7511ABSTRACT Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms. IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.Kurt SelleJoshua R. FletcherHannah TusonDaniel S. SchmittLana McMillanGowrinarayani S. VridhambalAlissa J. RiveraStephanie A. MontgomeryLouis-Charles FortierRodolphe BarrangouCasey M. TheriotDavid G. OusteroutAmerican Society for MicrobiologyarticleClostridioides difficileCRISPR-CasCas3phagelysogenyCRISPRMicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridioides difficile CRISPR-Cas Cas3 phage lysogeny CRISPR Microbiology QR1-502
spellingShingle	Clostridioides difficile CRISPR-Cas Cas3 phage lysogeny CRISPR Microbiology QR1-502 Kurt Selle Joshua R. Fletcher Hannah Tuson Daniel S. Schmitt Lana McMillan Gowrinarayani S. Vridhambal Alissa J. Rivera Stephanie A. Montgomery Louis-Charles Fortier Rodolphe Barrangou Casey M. Theriot David G. Ousterout <italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
description	ABSTRACT Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms. IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.
format	article
author	Kurt Selle Joshua R. Fletcher Hannah Tuson Daniel S. Schmitt Lana McMillan Gowrinarayani S. Vridhambal Alissa J. Rivera Stephanie A. Montgomery Louis-Charles Fortier Rodolphe Barrangou Casey M. Theriot David G. Ousterout
author_facet	Kurt Selle Joshua R. Fletcher Hannah Tuson Daniel S. Schmitt Lana McMillan Gowrinarayani S. Vridhambal Alissa J. Rivera Stephanie A. Montgomery Louis-Charles Fortier Rodolphe Barrangou Casey M. Theriot David G. Ousterout
author_sort	Kurt Selle
title	<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
title_short	<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
title_full	<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
title_fullStr	<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
title_full_unstemmed	<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials
title_sort	<italic toggle="yes">in vivo</italic> targeting of <named-content content-type="genus-species">clostridioides difficile</named-content> using phage-delivered crispr-cas3 antimicrobials
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/05d3dd5ddad5489e8af88e62d8ba3612
work_keys_str_mv	AT kurtselle italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT joshuarfletcher italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT hannahtuson italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT danielsschmitt italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT lanamcmillan italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT gowrinarayanisvridhambal italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT alissajrivera italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT stephanieamontgomery italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT louischarlesfortier italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT rodolphebarrangou italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT caseymtheriot italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials AT davidgousterout italictoggleyesinvivoitalictargetingofnamedcontentcontenttypegenusspeciesclostridioidesdifficilenamedcontentusingphagedeliveredcrisprcas3antimicrobials
_version_	1718427008992542720

<italic toggle="yes">In Vivo</italic> Targeting of <named-content content-type="genus-species">Clostridioides difficile</named-content> Using Phage-Delivered CRISPR-Cas3 Antimicrobials

Ejemplares similares