Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>

ABSTRACT The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated....

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Autores principales:	Kensuke Shima, Mary M. Weber, Christiane Schnee, Konrad Sachse, Nadja Käding, Matthias Klinger, Jan Rupp
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Chlamydia psittaci Gram-negative bacteria intracellular bacteria plasmid shuttle vector transformation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/fdf2345740494fec855eb256be0c90e7
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spelling	oai:doaj.org-article:fdf2345740494fec855eb256be0c90e72021-11-15T15:30:51ZDevelopment of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>10.1128/mSphere.00787-202379-5042https://doaj.org/article/fdf2345740494fec855eb256be0c90e72020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00787-20https://doaj.org/toc/2379-5042ABSTRACT The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions. IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci. In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci. We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci. Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.Kensuke ShimaMary M. WeberChristiane SchneeKonrad SachseNadja KädingMatthias KlingerJan RuppAmerican Society for MicrobiologyarticleChlamydia psittaciGram-negative bacteriaintracellular bacteriaplasmid shuttle vectortransformationMicrobiologyQR1-502ENmSphere, Vol 5, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Chlamydia psittaci Gram-negative bacteria intracellular bacteria plasmid shuttle vector transformation Microbiology QR1-502
spellingShingle	Chlamydia psittaci Gram-negative bacteria intracellular bacteria plasmid shuttle vector transformation Microbiology QR1-502 Kensuke Shima Mary M. Weber Christiane Schnee Konrad Sachse Nadja Käding Matthias Klinger Jan Rupp Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
description	ABSTRACT The obligate intracellular bacterium Chlamydia psittaci is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, C. psittaci can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in C. psittaci infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of C. psittaci using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian C. psittaci strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry includes genes encoding the green fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Target genes can be inserted at a multiple cloning site (MCS). Importantly, these genes can be regulated by a tetracycline-inducible (tet) promoter. Using the pCps-Tet-mCherry plasmid shuttle vector, we show the expression of GFP, as well as the induction of mCherry expression, in C. psittaci strain 02DC15, which belongs to the avian C. psittaci 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions. IMPORTANCE Psittacosis, caused by avian C. psittaci, has a major economic impact in the poultry industry worldwide and represents a significant risk for zoonotic transmission to humans. In the past decade, the tools of genetic manipulation have been improved for chlamydial molecular studies. While several genetic tools have been mainly developed in Chlamydia trachomatis, a stable gene-inducible shuttle vector system has not to date been available for C. psittaci. In this study, we adapted a C. trachomatis plasmid shuttle vector system to C. psittaci. We constructed a C. psittaci plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in C. psittaci. Importantly, exogeneous genes can be inserted at an MCS and are regulated by a tet promoter. The application of the pCps-Tet-mCherry shuttle vector system enables a promising new approach to investigate unknown gene functions of this pathogen.
format	article
author	Kensuke Shima Mary M. Weber Christiane Schnee Konrad Sachse Nadja Käding Matthias Klinger Jan Rupp
author_facet	Kensuke Shima Mary M. Weber Christiane Schnee Konrad Sachse Nadja Käding Matthias Klinger Jan Rupp
author_sort	Kensuke Shima
title	Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
title_short	Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
title_full	Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
title_fullStr	Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
title_full_unstemmed	Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>
title_sort	development of a plasmid shuttle vector system for genetic manipulation of <named-content content-type="genus-species">chlamydia psittaci</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/fdf2345740494fec855eb256be0c90e7
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Development of a Plasmid Shuttle Vector System for Genetic Manipulation of <named-content content-type="genus-species">Chlamydia psittaci</named-content>

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