Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrAHp) belongs to the well conserved family of serine proteases. HtrAHp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrAHp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrAHp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrAHp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrAHp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrAHp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrAHp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope

Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically

Effect of a Multi-Strain Probiotic Supplement on Gastrointestinal Symptoms and Serum Biochemical Parameters of Long-Distance Runners: A Randomized Controlled Trial

As many as 70% of athletes who practice endurance sports report experiencing gastrointestinal (GI) symptoms, such as abdominal pain, intestinal gurgling or splashing (borborygmus), diarrhea or the presence of blood in the stool, that occur during or after intense physical exercise. The aim of the study was to evaluate the effect of a multi-strain probiotic on the incidence of gastrointestinal symptoms and selected biochemical parameters in the serum of long-distance runners. After a 3-month intervention with a multi-strain probiotic, a high percentage of runners reported subjective improvement in their general health. Moreover, a lower incidence of constipation was observed. In the group of women using the probiotic, a statistically significant (p = 0.035) increase in serum HDL cholesterol concentration and a favorable lower concentration of LDL cholesterol and triglycerides were observed. These changes were not observed in the group of men using the probiotic. Probiotic therapy may reduce the incidence and severity of selected gastrointestinal symptoms in long-distance runners and improve subjectively assessed health condition

Chemical Composition of Winter Rape Seeds Depending on the Biostimulators Used

Plant growth regulators may reduce the negative effect of environmental stress factors and can contribute to increasing the quality and quantity of the yield. The aim of the research was to determine the effect of biostimulators on the quality of seeds of three winter rape morphotypes. Three varieties of winter rape were used: Poznaniak (population variety), PX104 (hybrid variety restored with a semi-dwarf growth type) and Konkret (hybrid variety restored with a traditional growth type). The varieties were exposed to three treatments: the biostimulator Tytanit®, the biostimulator Asahi®SL and the biostimulator Silvit®, and the control with no biostimulators. Seeds were analysed for content of crude fat, total fat and crude fibres. The biostimulators reduced total protein content (on average from 0.8 to 1.75 g·kg−1 of d.m.) and increased the concentration of crude fat (on average from 0.71 to 1.93 g·kg−1 of d.m.) and crude fibre (on average from 0.15 to 0.84 g·kg−1 of d.m.) compared to the control. PX104 had the highest content of crude fat and total fat protein, and the lowest in crude fibre. The smallest protein content was found in seeds of the long-stem hybrid Konkret, while crude fat was lowest in the population form (Poznaniak), and crude fibre was lowest in long-stem hybrid (Konkret)

Protective bacterial cultures and their use for inhibition of growth of listeriamonocytogenes in meat and meat products.

Liczne badania dowodzą, że mięso i produkty mięsne są źródłem chorobotwórczych bakterii Listeria monocytogenes. Potrzebne są różne strategie do zabezpieczenia produktu finalnego, aby zapobiec zakażeniu konsumentów. W pracy scharakteryzowano pałeczki Listeria monocytogenes oraz kultury ochronne stosowane do hamowania ich wzrostu. Zwrócono również uwagę na możliwość praktycznego wykorzystania bakterii mlekowych i wydzielanych przez nie bakteriocyn.Numerous investigations have provided evidence that meat and meat products are the source of pathological bacteria Listeria monocytogenes. Different strategies need to be applied in order to prevent consumers from contamination. In this review article, Listeria monocytogenes rods, and protective cultures of lactic acid bacteria ableto inhibit their growth are characterized. The attention is also paid to the possibilityof practical application of lactic acid bacteria and secreted by them bacteriocins

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions

Background Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. Methods The in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of β-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. Results We demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of β-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage. Conclusion Here we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen

Analysis of the Impact of a Multi-Strain Probiotic on Body Composition and Cardiorespiratory Fitness in Long-Distance Runners

Use of probiotic supplements, the benefits of which have not been proven in sportspeople, is becoming more widespread among runners. The aim of this study was to evaluate the effect of a multi-strain probiotic on body composition, cardiorespiratory fitness and inflammation in the body. The randomised, double-blind study included 66 long-distance runners. The intervention factor was a multi-strain probiotic or placebo. At the initial and final stages of the study, evaluation of body composition and cardiorespiratory fitness was performed and the presence of inflammation determined. In the group of men using the probiotic, an increase in lean body mass (p = 0.019) and skeletal muscle mass (p = 0.022) was demonstrated, while in the group of women taking the probiotic, a decrease in the content of total body fat (p = 0.600) and visceral fat (p = 0.247) was observed. Maximum oxygen consumption (VO2max) increased in women (p = 0.140) and men (p = 0.017) using the probiotic. Concentration of tumour necrosis factor-alpha decreased in women (p = 0.003) and men (p = 0.001) using the probiotic and in women (p = 0.074) and men (p = 0.016) using the placebo. Probiotic therapy had a positive effect on selected parameters of body composition and cardiorespiratory fitness of study participants and showed a tendency to reduce inflammation

Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA

Abstract Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81–176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells

E-Cadherin Orthologues as Substrates for the Serine Protease High Temperature Requirement A (HtrA)

Helicobacter pylori (H. pylori) expresses the serine protease and chaperone High temperature requirement A (HtrA) that is involved in periplasmic unfolded protein stress response. Additionally, H. pylori-secreted HtrA directly cleaves the human cell adhesion molecule E-cadherin leading to a local disruption of intercellular adhesions during pathogenesis. HtrA-mediated E-cadherin cleavage has been observed in response to a broad range of pathogens, implying that it is a prevalent mechanism in humans. However, less is known whether E-cadherin orthologues serve as substrates for bacterial HtrA. Here, we compared HtrA-mediated cleavage of human E-cadherin with murine, canine, and simian E-cadherin in vitro and during bacterial infection. We found that HtrA targeted mouse and dog E-cadherin equally well, whereas macaque E-cadherin was less fragmented in vitro. We stably re-expressed orthologous E-cadherin (Cdh1) in a CRISPR/Cas9-mediated cdh1 knockout cell line to investigate E-cadherin shedding upon infection using H. pylori wildtype, an isogenic htrA deletion mutant, or complemented mutants as bacterial paradigms. In Western blot analyses and super-resolution microscopy, we demonstrated that H. pylori efficiently cleaved E-cadherin orthologues in an HtrA-dependent manner. These data extend previous knowledge to HtrA-mediated E-cadherin release in mammals, which may shed new light on bacterial infections in non-human organisms

Biochemical properties of the HtrA homolog from bacterium Stenotrophomonas maltophilia

The HtrA proteins due to their proteolytic, and in many cases chaperone activity, efficiently counteract consequences of stressful conditions. In the environmental bacterium and nosocomial pathogen Stenotrophomonas maltophilia HtrA (HtrA.Sm) is induced as a part of adaptive response to host temperature (37. \uc2\ub0C).We examined the biochemical properties of HtrA.Smand compared them with those of model HtrA.Ecfrom Escherichia coli. We found that HtrA.Smis a protease and chaperone that operates over a wide range of pH and is highly active at temperatures between 35 and 37. \uc2\ub0C. The temperature-sensitive activity corresponded well with the lower thermal stability of the protein and weaker stability of the oligomer. Interestingly, the enzyme shows slightly different substrate cleavage specificity when compared to other bacterial HtrAs. A computational model of the three-dimensional structure of HtrA.Smindicates differences in the S1 substrate specificity pocket and suggests weaker inter-trimer interactions when compared to HtrA.Ec.The observed features of HtrA.Smsuggest that this protein may play a protective role under stressful conditions acting both as a protease and a chaperone. The optimal temperatures for the protein activity may reflect the evolutionary adaptation of S. maltophilia to life in soil or aqueous environments, where the temperatures are usually much below 37. \uc2\ub0C