HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Design, synthesis and biological evaluation of P2-modified proline analogues targeting the HtrA serine protease in Chlamydia.

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 1,2,3-triazole 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.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues.

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C. trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50 µM) in C. trachomatis and C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.

A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro Evidence of Incorporation.

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.

Oxidoreductase disulfide bond proteins DsbA and DsbB form an active redox pair in Chlamydia trachomatis, a bacterium with disulfide dependent infection and development.

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.

Terminomics Methodologies and the Completeness of Reductive Dimethylation: A Meta-Analysis of Publicly Available Datasets.

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.