The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause specific diseases, it is imperative to develop a mutagenesis approach that would be applicable to every chlamydial species. We present functional evidence that the region between Chlamydia trachomatis and Chlamydia muridarum pgp6 and pgp7, containing four 22-bp tandem repeats that are present in all chlamydial endogenous plasmids, represents the plasmid origin of replication. Furthermore, by introducing species-specific ori regions into an engineered 5.45-kb pUC19-based plasmid, we generated vectors that can be successfully transformed into and propagated under selective pressure by C. trachomatis serovars L2 and D, as well as C. muridarum. Conversely, these vectors were rapidly lost upon removal of the selective antibiotic. This conditionally replicating system was used to generate a tarP deletion mutant by fluorescence-reported allelic exchange mutagenesis in both C. trachomatis serovar D and C. muridarum. The strains were analyzed using in vitro invasion and fitness assays. The virulence of the C. muridarum strains was then assessed in a murine infection model. Our approach represents a novel and efficient strategy for targeted genetic manipulation in Chlamydia beyond C. trachomatis L2. This advance will support comparative studies of species-specific infection biology and enable studies in a well-established murine model of chlamydial pathogenesis.
|Journal||Infection and Immunity|
|State||Published - Dec 2022|
Bibliographical noteFunding Information:
This study was supported by National Institutes of Health (NIH) grants R35 GM144123-01 (to T.R.G.) and 1R01AI53599-01 (to T.B.R.). The VUMC Flow Cytometry Shared Resource is supported by NIH grants to the Vanderbilt Ingram Cancer Center (P30-CA68485) and the Vanderbilt Digestive Disease Research Center (P30-DK058404). The content here is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We declare no competing interests.
© 2022 American Society for Microbiology.
ASJC Scopus subject areas
- Infectious Diseases