Investigating the role of the HtrA protease in chlamydial biology and the therapeutic potential of a HtrA inhibitor

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Chlamydia are small, obligate intracellular bacteria that are able to infect a wide variety of hosts, including humans, livestock, and domestic species. Chlamydial infections cause significant morbidity in humans and animals alike, with substantial impacts on public health, veterinary health, and agriculture. Despite the high prevalence of infections and associated morbidities, treatment options are limited with several barriers to use, such as antibiotic resistance in other prevalent sexually transmitted bacteria and the disruption of gut microflora in koalas. JO146 is a novel anti-chlamydial drug candidate and inhibitor of HtrA, a serine protease that is crucial for the development of Chlamydia. Importantly, JO146 may be selective for chlamydial HtrA, and it is currently the only drug candidate with demonstrated efficacy against the key koala pathogen Chlamydia pecorum. Mutant isolates of the human pathogen Chlamydia trachomatis with acquired resistance to JO146 developed unexpected single nucleotide variations within fatty acid biosynthesis pathways, presenting a unique opportunity to further explore chlamydial biology and a potential link between HtrA and fatty acid metabolism. This project aimed to characterise JO146-resistant C. trachomatis mutants and continue optimisation of JO146 as a novel drug candidate. Investigation of C. trachomatis JO146-resistant mutant phenotypes in vitro indicated that the isolates were fit, with subtle yet significant differences in infection dynamics compared to the wild-type parent strain. Molecular characterisation revealed distinct lipid profiles in the mutant isolates relative to wild-type that were associated with an increased abundance of HtrA and the outer membrane proteins MOMP and PmpD. Mutant isolates also demonstrated equivalent permeability to JO146 as the wild-type, indicating that the mechanism of JO146 resistance was likely directly related to the distinct lipid profiles and increased levels of HtrA. Importantly, novel polyunsaturated fatty acid species not previously detected in C. trachomatis cultures were also identified. Several novel JO146 analogues demonstrated improved activity against HtrA, with efficacy against C. trachomatis and C. pecorum. Specifically, replacement of valine with a tertiary leucine at the P3 position and acetate ester modification of proline at P2 were determined to improve drug activity. This project has identified a novel interplay between fatty acid and phospholipid metabolism, HtrA, and the membrane in C. trachomatis, and further optimised JO146 as a specific, novel anti-chlamydial drug.
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