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bioRxiv · 10.1101/2024.02.22.581593

Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development in Chlamydia trachomatis

Junker, S.·Singh, V.·Al-Saadi, A. G. M.·Wood, N. A.·Hamilton-Brehm, S. D.·Ouellette, S. P.·Fisher, D. J.
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Abstract

Partner Switching Mechanisms (PSM) are signal transduction systems comprised of a sensor phosphatase (RsbU), an anti-sigma factor (RsbW, kinase), an anti-anti-sigma factor (RsbV, the RsbW substrate), and a target sigma factor. Chlamydia spp. are obligate intracellular bacterial pathogens of animals that undergo a developmental cycle transitioning between the infectious elementary body (EB) and replicative reticulate body (RB) within a host-cell derived vacuole (inclusion). Secondary differentiation events (RB to EB) are transcriptionally regulated, in part, by the house-keeping sigma factor ({sigma}66) and two late-gene sigma factors ({sigma}54 and {sigma}28). Prior research supports that the PSM in Chlamydia trachomatis regulates availability of {sigma}66. Pan-genome analysis revealed that PSM components are conserved across the phylum Chlamydiota, with Chlamydia spp. possessing an atypical arrangement of two anti-anti-sigma factors, RsbV1 and RsbV2. Bioinformatic analyses support RsbV2 as the homolog to the pan-genome conserved RsbV with RsbV1 as an outlier. This, combined with in vitro data, indicates that RsbV1 and RsbV2 are structurally and biochemically distinct. Reduced levels or overexpression of RsbV1/RsbV2 did not significantly impact C. trachomatis growth or development. In contrast, overexpression of a non-phosphorylatable RsbV2 S55A mutant, but not overexpression of an RsbV1 S56A mutant, resulted in a 3 log reduction in infectious EB production without reduction in genomic DNA (total bacteria) or inclusion size, suggesting a block in secondary differentiation. The block was corroborated by reduced production of {sigma}54/28-regulated late proteins and via transmission electron microscopy. ImportanceC. trachomatis is the leading cause of reportable bacterial sexually transmitted infections (STIs) and causes the eye infection trachoma, a neglected tropical disease. Broad-spectrum antibiotics used for treatment can lead to microbiome dysbiosis and increased antibiotic resistance development in other bacteria, and treatment failure for chlamydial STIs is a recognized clinical problem. Here, we show that disruption of a partner switching mechanism (PSM) significantly reduces infectious progeny production via blockage of RB to EB differentiation. We also reveal a novel PSM expansion largely restricted to the species infecting animals, suggesting a role in pathogen evolution. Collectively, our results highlight the chlamydial PSM as a key regulator of development and as a potential target for the development of novel therapeutics to treat infections.

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Junker, S., Singh, V., Al-Saadi, A. G. M., Wood, N. A., Hamilton-Brehm, S. D., Ouellette, S. P., Fisher, D. J.. 2024-02-23. Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development in Chlamydia trachomatis. https://doi.org/10.1101/2024.02.22.581593

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