No abstract

High temperature requirement A (HtrA) serine proteases have emerged as a novel class of antibacterial target, which are crucial in protein quality control and are involved in the pathogenesis of a wide array of bacterial infections. Previously, we demonstrated that HtrA in Chlamydia is essential for bacterial survival, replication and virulence. Here, we report a new series of proline (P2)-modified inhibitors of Chlamydia trachomatis HtrA (CtHtrA) developed by proline ring expansion and Cγ-substitutions. The structure-based drug optimization process was guided by molecular modelling and in vitro pharmacological evaluation of inhibitory potency, selectivity and cytotoxicity. Compound 25 from the first-generation 4-substituted proline analogues increased antiCtHtrA potency and selectivity over human neutrophil elastase (HNE) by approximately 6-and 12-fold, respectively, relative to the peptidic lead compound 1. Based on this compound, second-generation substituted proline residues containing 1,2,3triazole moieties were synthesized by regioselective azide-alkyne click chemistry. Compound 49 demonstrated significantly improved antichlamydial activity in whole cell assays, diminishing the bacterial infectious progeny below the detection limit at the lowest dose tested. Compound 49 resulted in approximately 9-and 22-fold improvement in the inhibitory potency and selectivity relative to 1, respectively. To date, compound 49 is the most potent HtrA inhibitor developed against Chlamydia spp. a Relative inhibition against CtHtrA is the ratio of IC50 CtHtrA(1) to IC50 CtHtrA (compound).b Relative inhibition against HNE is the ratio of IC50 HNE (1) to IC50 HNE (compound).

Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and—using recombinant purified proteins—demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein’s thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle.

Methods for analyzing the terminal sequences of proteins have been refined over the previous decade; however, few studies have evaluated the quality of the data that have been produced from those methodologies. While performing global N-terminal labelling on bacteria, we observed that the labelling was not complete and investigated whether this was a common occurrence. We assessed the completeness of labelling in a selection of existing, publicly available N-terminomics datasets and empirically determined that amine-based labelling chemistry does not achieve complete labelling and potentially has issues with labelling amine groups at sequence-specific residues. This finding led us to conduct a thorough review of the historical literature that showed that this is not an unexpected finding, with numerous publications reporting incomplete labelling. These findings have implications for the quantitation of N-terminal peptides and the biological interpretations of these data.

Proteinopathies are protein misfolding diseases that have an underlying factor that affects the conformation of proteoforms. A factor hypothesised to play a role in these diseases is the incorporation of non-protein amino acids into proteins, with a key example being the therapeutic drug levodopa. The presence of levodopa as a protein constituent has been explored in several studies, but it has not been examined in a global proteomic manner. This paper provides a proof-of-concept method for enzymatically creating levodopa-containing proteins using the enzyme tyrosinase and provides spectral evidence of in vitro incorporation in addition to the induction of the unfolded protein response due to levodopa.