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

The obligate intracellular bacterium is a known avian pathogen causing psittacosis in birds and is capable of zoonotic transmission. In human pulmonary infections, can cause pneumonia associated with significant mortality if inadequately diagnosed and treated. Although intracellular manipulates host cell organelles for its replication and survival, it has been difficult to demonstrate host-pathogen interactions in infection due to the lack of easy-to-handle genetic manipulation tools. Here, we show the genetic transformation of using a plasmid shuttle vector that contains a controllable gene induction system. The 7,553-bp plasmid p01DC12 was prepared from the nonavian strain 01DC12. We constructed the shuttle vector pCps-Tet-mCherry using the full sequence of p01DC12 and the 4,449-bp fragment of 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 strain 02DC15, which belongs to the avian 6BC clade. Furthermore, we demonstrated that pCps-Tet-mCherry was stably retained in transformants. Thus, our plasmid shuttle vector system represents a novel targeted approach that enables the elucidation of host-pathogen interactions. Psittacosis, caused by avian , 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 , a stable gene-inducible shuttle vector system has not to date been available for In this study, we adapted a plasmid shuttle vector system to We constructed a plasmid backbone shuttle vector called pCps-Tet-mCherry. The construct expresses GFP in 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.