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Research Article | Host-Microbe Biology

Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

Kensuke Shima, Mary M. Weber, Christiane Schnee, Konrad Sachse, Nadja Käding, Matthias Klinger, Jan Rupp
Sarah E. F. D'Orazio, Editor
Kensuke Shima
aDepartment of Infectious Diseases and Microbiology, University of Lübeck, Lübeck, Germany
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Mary M. Weber
bDepartment of Microbiology and Immunology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA
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Christiane Schnee
cInstitute of Molecular Pathogenesis, Friedrich-Loeffler-lnstitut (Federal Research Institute for Animal Health), Jena, Germany
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Konrad Sachse
dRNA Bioinformatics and High-Throughput Analysis, Faculty of Mathematics and Computer Science, Friedrich-Schiller-Universität Jena, Jena, Germany
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Nadja Käding
aDepartment of Infectious Diseases and Microbiology, University of Lübeck, Lübeck, Germany
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Matthias Klinger
eInstitute of Anatomy, University of Lübeck, Lübeck, Germany
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Jan Rupp
aDepartment of Infectious Diseases and Microbiology, University of Lübeck, Lübeck, Germany
fGerman Center for Infection Research (DZIF), partner site Hamburg-Lübeck-Borstel, Germany
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Sarah E. F. D'Orazio
University of Kentucky
Roles: Editor
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DOI: 10.1128/mSphere.00787-20
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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.

  • Copyright © 2020 Shima et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci
Kensuke Shima, Mary M. Weber, Christiane Schnee, Konrad Sachse, Nadja Käding, Matthias Klinger, Jan Rupp
mSphere Aug 2020, 5 (4) e00787-20; DOI: 10.1128/mSphere.00787-20

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Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci
Kensuke Shima, Mary M. Weber, Christiane Schnee, Konrad Sachse, Nadja Käding, Matthias Klinger, Jan Rupp
mSphere Aug 2020, 5 (4) e00787-20; DOI: 10.1128/mSphere.00787-20
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KEYWORDS

Chlamydia psittaci
Gram-negative bacteria
intracellular bacteria
plasmid shuttle vector
transformation

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