Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to <i><scp>C</scp>hlamydia trachomatis</i>

Gloeckl, Sarina; Ong, Vanissa A.; Patel, Pooja; Tyndall, Joel D. A.; Timms, Peter; Beagley, Kenneth W.; Allan, John; Armitage, Charles W.; Turnbull, Lynne; Whitchurch, Cynthia B.; Merdanovic, Melisa; Ehrmann, Michael; Powers, James C.; Oleksyszyn, Józef; Verdoes, Martijn; Bogyo, Matthew; Huston, Wilhelmina M.

doi:10.1111/mmi.12306

Cited by 52 publications

(84 citation statements)

References 28 publications

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“…In H. pylori or C. trachomatis , genomic htrA deletions mutants could not be generated so far. However, functional small molecule inhibitors and substrate-derived peptide inhibitors were designed which efficiently blocked HtrA functions [30, 33, 34]. HtrA-mediated E-cadherin cleavage was also shown for EPEC and S. flexneri supporting our hypothesis that E-cadherin ectodomain shedding might be a prevalent mechanism for pathogenic bacteria to promote virulence through the interference with (baso-) lateral domains of epithelial cells [15].…”

Section: Introductionmentioning

confidence: 93%

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens

et al. 2016

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BackgroundThe serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function.MethodsE-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine—to—alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments.ResultsInfection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro.ConclusionsThese data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-016-0153-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 93%

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens

et al. 2016

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“…In addition, it has been reported that HtrA is crucial for the virulence of numerous bacterial pathogens such as Yersinia enterocolitica [41], Salmonella enterica [42], Klebsiella pneumonia [43], Shigella fl exneri [44], Listeria monocytogenes [45], and Chlamydia trachomatis [46]. It is wellknown that HtrA is critical for stress tolerance and survival of most bacteria, because it degrades and prevents aggregation of periplasmic proteins that can misfold under stress conditions [31-33], but only a few reports have studied the discrete functions of HtrA protease and chaperone activities during infection [27-30].…”

Section: Discussionmentioning

confidence: 99%

Campylobacter jejuniserine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

Boehm¹,

Lind²,

Backert³

et al. 2015

European Journal of Microbiology and Immunology

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Campylobacter jejuni is an important pathogen of foodborne illness. Transmigration across the intestinal epithelial barrier and invasion are considered as primary reasons for tissue damage triggered by C. jejuni. Using knockout mutants, it was shown that the serine protease HtrA may be important for stress tolerance and physiology of C. jejuni. HtrA is also secreted in the extracellular environment, where it can cleave junctional host cell proteins such as E-cadherin. Aim of the present study was to establish a genetic complementation system in two C. jejuni strains in order to introduce the wild-type htrA gene in trans, test known htrA phenotypes, and provide the basis to perform further mutagenesis. We confirm that reexpression of the htrA wild-type gene in ΔhtrA mutants restored the following phenotypes: 1) C. jejuni growth at high temperature (44 °C), 2) growth under high oxygen stress conditions, 3) expression of proteolytically active HtrA oligomers, 4) secretion of HtrA into the supernatant, 5) cell attachment and invasion, and 6) transmigration across polarized epithelial cells. These results establish a genetic complementation system for htrA in C. jejuni, exclude polar effects in the ΔhtrA mutants, confirm important HtrA properties, and permit the discovery and dissection of new functions.

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“…Activitybased probes have been designed to detect a number of bacterial proteins, such as sulfatases 85,86 , serine proteases 87 , the cell wall biosynthetic protein MurA 88 , glycoside hydrolases 89 , redox-sensitive proteins 90,91 and histidine kinases 92 ; however, most of these probes have been used only in gel-based analyses. One additional strategy that has been applied to bacterial imaging is the use of a probe that becomes fluorescent upon turnover by nitroreductase.…”

Section: Additional Small-molecule Probesmentioning

confidence: 99%

Progress and prospects for small-molecule probes of bacterial imaging

Kocaoglu

Carlson

2016

Nat Chem Biol

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Fluorescence microscopy is an essential tool for the exploration of cell growth, division, transcription and translation in eukaryotes and prokaryotes alike. Despite the rapid development of techniques to study bacteria, the size of these organisms (1–10 μm) and their robust and largely impenetrable cell envelope present major challenges in imaging experiments. Fusion-based strategies, such as attachment of the protein of interest to a fluorescent protein or epitope tag, are by far the most common means for examining protein localization and expression in prokaryotes. While valuable, the use of genetically encoded tags can result in mislocalization or altered activity of the desired protein, does not provide a readout of the catalytic state of enzymes and cannot enable visualization of many other important cellular components, such as peptidoglycan, lipids, nucleic acids or glycans. Here, we highlight the use of biomolecule-specific small-molecule probes for imaging in bacteria.

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Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis

Cited by 52 publications

References 28 publications

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens

Campylobacter jejuniserine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration

Progress and prospects for small-molecule probes of bacterial imaging

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