Generation of deletion mutants in any chlamydial species: Quick and easy!

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Abstract Members of the genus Chlamydia are Gram-negative bacteria that cause various acute as well as chronic diseases in humans. Although chlamydiae share numerous aspects of their typical bacterial life style, such as intracellular developmental cycle and the preference to primarily invade epithelial cells, these human pathogens employ sophisticated species-specific strategies contributing to an extraordinary diversity in tissue tropism and disease manifestation. C. trachomatis infects mucosal surfaces of the human urogenital tract or columnar epithelial cells of the eye. Chlamydia-mediated genital infections represent the leading cause of bacterial STDs in the United States, with 1, 758,668 infections reported to the CDC in 2018. C. trachomatis isolates are divided into 15 major serovariants. The less invasive isolates, which cause infections of mucosal layers, include ocular serovars A, B, Ba, C, while serovars D through K cause sexually transmitted diseases. The lymphogranuloma venereum (LGV) serovars, L1, L2, and L3, transiently infect epithelial cells but then invade the submucosae to infect macrophages, which facilitates the dissemination of the bacteria to the regional lymph nodes. Genetic manipulation of C. trachomatis, L2, which has been extensively developed in the last decade, significantly helped to elucidate how these pathogens interact with their hosts and possibly, cause diseases. However, in order to discover and understand the mechanism(s) for chlamydia- specific host tropism and pathogenesis outcomes with each chlamydial species, it is imperative to expend molecular genetics technologies to all chlamydial species and strains. We propose here, to optimize a unique, recently developed approach for generation of null mutants by allelic exchange via homologous recombination, using pUC19 shuttle vector, that would be applicable to any member of the genus Chlamydia. We intend to utilize this approach in production of deletion mutants in pmpI and pmpD in C. trachomatis, serovars L2, D, and B. Moreover, the pmpI and pmpD will be knocked out in C. mudirarum since this rodent chlamydial species is typically employed in studies investigating chlamydial pathogenesis in vivo. Phenotypical studies with ∆pmpI and ∆pmpD chlamydiae with their WT counterparts and complemented strains will be conducted in various human as well as murine cells cultures in order to identify possible species-specific and host-specific interactions.