The Genetic Transformation of Chlamydia pneumoniae

Abstract

We demonstrate the genetic transformation of <i>Chlamydia pneumoniae</i> using a plasmid shuttle vector system which generates stable transformants. The equine <i>C. pneumoniae</i> N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing <i>C. pneumoniae</i> transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate <i>C. pneumoniae</i> LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate <i>C. pneumoniae</i> CV-6 and the human community-acquired pneumonia-associated <i>C. pneumoniae</i> IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that <i>Chlamydia</i> spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free <i>C. trachomatis</i>, <i>C. muridarum</i>, <i>C. caviae</i>, <i>C. pecorum</i>, and <i>C. abortus</i> However, contrary to our expectation, pRSGFPCAT-Cpn did transform <i>C. felis</i> Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of <i>C. felis</i> Taken together, we provide for the first time an easy-to-handle transformation protocol for <i>C. pneumoniae</i> that results in stable transformants. In addition, the vector can cross the species barrier to <i>C. felis</i>, indicating the potential of horizontal pathogenic gene transfer via a plasmid.<b>IMPORTANCE</b> The absence of tools for the genetic manipulation of <i>C. pneumoniae</i> has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for <i>C. pneumoniae</i> transformation based on a plasmid shuttle vector system. We constructed a <i>C. pneumoniae</i> plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in <i>C. pneumoniae</i><i>C. pneumoniae</i> transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in <i>C. pneumoniae</i> using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in <i>C. pneumoniae</i> biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.