htrA Mutation Research Articles

A single-nucleotide polymorphism in Helicobacter pylori promotes gastric cancer development

Single-nucleotide polymorphisms (SNPs) in various human genes are key factors in carcinogenesis. However, whether SNPs in bacterial pathogens are similarly crucial in cancer development is unknown. Here, we analyzed 1,043 genomes of the stomach pathogen Helicobacter pylori and pinpointed a SNPin the serine protease HtrA (position serine/leucine 171) that significantly correlates with gastric cancer. Our functional studies reveal that the 171S-to-171L mutation triggers HtrA trimer formation and enhances proteolytic activity and cleavage of epithelial junction proteins occludin and tumor-suppressor E-cadherin. 171L-type HtrA, but not 171S-HtrA-possessing H.pylori, inflicts severe epithelial damage, enhances injection of oncoprotein CagA into epithelial cells, increases NF-κB-mediated inflammation and cell proliferation through nuclear accumulation of β-catenin, and promotes host DNA double-strand breaks, collectively triggering malignant changes. These findings highlight the 171S/L HtrA mutation as a unique bacterial cancer-associated SNP and as a potential biomarker for risk predictions in H.pylori infections.

Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations.

High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the inter-residue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation─a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

HtrA of Actinobacillus pleuropneumoniae is a virulence factor that confers resistance to heat shock and oxidative stress

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, which is a severe and often fatal disease that results in significant economic loss. The means by which A. pleuropneumoniae survives within the host are not clear. High temperature requirement A (HtrA) proteases have been shown to affect cell viability during stressful conditions and are virulence factors in many bacterial species. In this study, we examined the biological role of HtrA during A. pleuropneumoniae infection by analyzing the impact of htrA mutation on virulence-associated phenotypes. We found that htrA mutation had a dramatic impact on stress tolerance. The htrA mutant (ΔhtrA) displayed a lethal phenotype at elevated temperature (42 °C). Further, ΔhtrA exhibited increased susceptibility to H2O2-induced oxidative stress when compared to the parental strain (SLW01) and a complementation strain (ΔhtrA-Compl). Animal infection assays demonstrated that absence of HtrA led to decreased in vivo colonization ability, and ΔhtrA is less virulent in pigs relative to SLW01. Furthermore, pig competitive infection assays demonstrated fewer blood associated CFUs with ΔhtrA infection than with SLW01. These results demonstrate HtrA plays a significant role in the survival and growth of A. pleuropneumoniae during stressful conditions, and that immune escape and invasiveness are important to the process of A. pleuropneumoniae infection.

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.

Role of HtrA in surface protein expression and biofilm formation by Streptococcus mutans.

The HtrA surface protease in gram-positive bacteria is involved in the processing and maturation of extracellular proteins and degradation of abnormal or misfolded proteins. Inactivation of htrA has been shown to affect the tolerance to thermal and environmental stress and to reduce virulence. We found that inactivation of Streptococcus mutans htrA by gene-replacement also resulted in a reduced ability to withstand exposure to low and high temperatures, low pH, and oxidative and DNA damaging agents. The htrA mutation affected surface expression of several extracellular proteins including glucan-binding protein B (GbpB), glucosyltransferases, and fructosyltransferase. In addition, htrA mutation also altered the surface expression of enolase and glyceraldehyde-3-phosphate dehydrogenease, two glycolytic enzymes that are known to be present on the streptococcal cell surface. As expected, microscopic analysis of in vitro grown biofilm structure revealed that the htrA deficient biofilms adopted a much more granular patchy appearance, rather than the relatively smooth confluent layer normally seen in the wild type. These results suggest that HtrA plays an important role in the biogenesis of extracellular proteins including surface associated glycolytic enzymes and in biofilm formation of S. mutans.

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91 phox −/− mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9l phox −/− mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9l phox −/− mice, with more than 60% reduction in cell numbers 2.5 h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9l phox −/− mice, whereas an aroA live vaccine strain had no effect on either normal or gp9l phox −/− macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.

Susceptibility of calves to challenge with Salmonella typhimurium 4/74 and derivatives harbouring mutations in htrA or purE.

Salmonella typhimurium 4/74 is highly virulent for cattle after oral challenge, causing severe diarrhoea, which is sometimes associated with systemic spread of the micro-organism. Although susceptible to oral challenge, groups of cattle were found to be relatively resistant to subcutaneous challenge with this strain. The virulence of S. typhimurium 4/74 harbouring mutations in htrA and purE was also assessed in cattle. Although S. typhimurium 4/74 htrA and purE are attenuated following oral challenge in mice, cattle were highly susceptible to oral challenge with these mutants. As with the parent S. typhimurium 4/74 strain, cattle exhibited greater susceptibility to oral compared to subcutaneous challenge with S. typhimurium htrA and purE mutants. Following subcutaneous challenge with sublethal levels of S. typhimurium 4/74, calves produced significant levels of antibodies to S. typhimurium soluble extract. No correlation was detected between interferon gamma levels in sera and susceptibility to infection by any route. The concentrations of the acute-phase-associated protein haptoglobin were increased in the sera of five of six cattle inoculated subcutaneously, although increases in concentration were smaller in cattle inoculated orally.

The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress.

The serine protease HtrA (DegP), which is indispensable for cell survival at elevated temperatures, is a peripheral membrane protein, localized on the periplasmic side of the inner membrane in Escherichia coli, and the biochemical and genetic evidence indicates that the physiological role of HtrA is to degrade denatured proteins formed in the cellular envelope during heat shock. The aim of this study was to find out if the HtrA protease contributes to protection of the cell against oxidative stress. We compared the influence of various oxidizing agents on htrA mutant cells with their effects on wild-type bacteria, and found that the htrA mutation did not increase sensitivity to hydrogen peroxide or paraquat but made the cell extremely sensitive to ferrous [Fe(II)] ions, which are known to enhance oxidation of proteins. Treatment with ferrous ions caused a larger increase in the level of protein carbonyl groups in the membrane fraction of the cell than in the periplasm and cytoplasm. Iron-induced oxidation of membrane proteins was enhanced in the htrA mutant relative to wild-type cells. Inhibition of the growth of the htrA mutant by iron could be alleviated more efficiently by a nitroxide antioxidant that localizes in the membranes (A-TEMPO) than by a derivative (40H-TEMPO) that acts mainly in the soluble fraction of the cell. Inhibition of the growth of the htrA mutant was more pronounced following treatment with cumene hydroperoxide, which partitions into membranes, than with t-butyl hydroperoxide, which forms radical mainly in the cytosol. Both ferrous ions and cumene hydroperoxide, but not hydrogen peroxide, paraquat or t-butyl hydroperoxide, induced synthesis of HtrA. Our results show that HtrA plays a role in defense against oxidative shock and support the hypothesis that HtrA participates in the degradation of oxidatively damaged proteins localized in the cell envelope, especially those associated with the membranes.

Characterization and genetic complementation of a Brucella abortus high-temperature-requirement A (htrA) deletion mutant.

In order to evaluate the biological function of the Brucella abortus high-temperature-requirement A (HtrA) stress response protein homolog, the majority of the htrA gene was deleted from the chromosome of B. abortus 2308 via gene replacement. In contrast to the parental strain, the resulting htrA deletion mutant, designated PHE1, failed to grow on solid medium at 40 degrees C and demonstrated increased sensitivity to killing by H2O2 and O2- in disk sensitivity assays. BALB/c mice were infected with strains 2308 and PHE1 to assess the effect of the htrA mutation on virulence, and significantly fewer brucellae were recovered from the spleens of mice infected with PHE1 than from those of mice infected with 2308 at 1 week postinfection. Genetic complementation studies were performed to confirm the relationship between the htrA mutation and the phenotype observed for PHE1. Plasmid pRIE1 was constructed by inserting a 1.9-kb EcoRI fragment encoding the B. abortus htrA gene into the broad-host-range plasmid pBBR1MCS. Introduction of pRIE1 into PHE1 relieved the temperature- and H2O2-sensitive phenotypes of this mutant in vitro, and PHE1(pRIE1) colonized the spleens of BALB/c mice at levels equivalent to those of the parental 2308 strain at 1 week postinfection. These results support our previous proposal that the B. abortus htrA gene product functions as a stress response protein and further suggest that this protein contributes to virulence. These studies also demonstrate the utility of the broad-host-range plasmid pBBR1MCS for genetic complementation studies in Brucella spp., establishing a key reagent for more detailed genetic analysis of this important zoonotic pathogen.

Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model

Derivatives of the mouse-virulent Salmonella typhimurium strain SL1344 were constructed harbouring defined mutations in htrA, aroA or htrA aroA combined. When administered orally or intravenously to BALB/c mice, all the mutants were found to be highly attenuated. All mutants were able to confer significant protection against lethal challenge with SL1344 after a single oral dose of live organisms. SL1344 htrA mutants persisted in livers and spleens at a lower level than SL1344 aroA mutants after intravenous administration. SL1344 htrA aroA mutants persisted at an even lower level and were cleared from the livers and spleens of mice within 21 days of intravenous administration. Thus htrA and htrA aroA mutants can be considered as potential oral vaccines against salmonellosis.