MAINTENANCE OF INFECTIVITY AND TARGETED GENETIC CONTROL OF REPLICATION AND MORPHOLOGICAL DEVELOPMENT IN CHLAMYDIA

Cameron Mandel

doi:10.7273/000004636

The mucosotropic, Gram-negative, bacterial obligate intracellular pathogen Chlamydia trachomatis is a leading cause of preventable blindness, and the most prevalent sexually transmitted bacterium in the United States. Up to 70% of infected individuals are asymptomatic carriers of C. trachomatis, capable of unknowingly transmitting the pathogen to susceptible individuals. Clinical infection can manifest as epididymitis in men and pelvic inflammatory disease or infertility in women. Trachoma, the ocular manifestation of disease, is characterized by recurrent inflammation of conjunctival tissues resulting in scarring and trichiasis if left untreated. Upon infection, C. trachomatis invades mucosal epithelial cells and replicates intracellularly via an asynchronous biphasic developmental cycle consisting of a vegetative but non-infectious Reticulate Body (RB) and an infectious, non-replicative Elementary Body (EB). Replication exclusively occurs inside of a membrane-bound compartment termed the chlamydial inclusion; however, recent studies have aimed to characterize EB maintenance of infectivity in the extracellular space prior to infection. A model for axenic incubation of EBs in natural mucosal secretions was developed in order to characterize EB maintenance of infectivity in a physiologically relevant substrate. EBs incubated in axenic buffer formulations conditioned with murine ocular secretions exhibited prolonged EB maintenance of infectivity compared to base buffers alone. Genetic determinants of C. trachomatis morphological transitions are poorly defined, but the genes involved are likely associated with transcriptional reprogramming of the pathogen. In Escherichia coli, the transcription factor DksA, and the transcription elongation factor GreA modulate RNA polymerase in response to nutrient availability. The chlamydial genome contains DksA and GreA orthologs (GreACt and DksACt) but lacks the upstream regulatory enzymes RelA/SpoT. Ectopic overexpression of DksACt resulted in a mild decrease in replication and a moderate decrease in infectivity. The crystal structure of the DksACt is consistent with canonical DksA structures. Ectopic overexpression of GreACt resulted in no change in replication and a marked decrease in infectivity. Images of affected bacteria obtained via transmission electron microscopy indicated that overexpression of GreACt results in a population of predominantly RBs, compared to a mixed population of RBs and EBs in non-treated samples. Data presented here indicate that GreACt interferes with RB-EB transitions.

MAINTENANCE OF INFECTIVITY AND TARGETED GENETIC CONTROL OF REPLICATION AND MORPHOLOGICAL DEVELOPMENT IN CHLAMYDIA

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