In vitro anti-Helicobacter pylori activity and antivirulence activity of cetylpyridinium chloride.

The primary treatment method for eradicating Helicobacter pylori (H. pylori) infection involves the use of antibiotic-based therapies. Due to the growing antibiotic resistance of H. pylori, there has been a surge of interest in exploring alternative therapies. Cetylpyridinium chloride (CPC) is a water-soluble and nonvolatile quaternary ammonium compound with exceptional broad-spectrum antibacterial properties. To date, there is no documented or described specific antibacterial action of CPC against H. pylori. Therefore, this study aimed to explore the in vitro activity of CPC against H. pylori and its potential antibacterial mechanism. CPC exhibited significant in vitro activity against H. pylori, with MICs ranging from 0.16 to 0.62 µg/mL and MBCs ranging from 0.31 to 1.24 µg/mL. CPC could result in morphological and physiological modifications in H. pylori, leading to the suppression of virulence and adherence genes expression, including flaA, flaB, babB, alpA, alpB, ureE, and ureF, and inhibition of urease activity. CPC has demonstrated in vitro activity against H. pylori by inhibiting its growth, inducing damage to the bacterial structure, reducing virulence and adherence factors expression, and inhibiting urease activity.

Substitution of Asp29 with Asn29 in the metallochaperone UreE of Streptococcus thermophilus DSM 20617T increases the urease activity and anticipates urea hydrolysis during milk fermentation.

Urease operon is highly conserved within the species Streptococcus thermophilus and urease-negative strains are rare in nature. S. thermophilus MIMO1, isolated from commercial yogurt, was previously characterized as urease-positive Ni-dependent strain. Beside a mutation in ureQ, coding for a nickel ABC transporter permease, the strain MIMO1 showed a mutation in ureE gene which code for a metallochaperone involved in Ni delivery to the urease catalytic site. The single base mutation in ureE determined a substitution of Asp29 with Asn29 in the metallochaperone in a conserved protein region not involved in the catalytic activity. With the aim to investigate the role Asp29vs Asn29 substitution in UreE on the urease activity of S. thermophilus, ureE gene of the reference strain DSM 20617T (ureEDSM20617) was replaced by ureE gene of strain MIMO1 (ureEMIMO1) to obtain the recombinant ES3. In-gel detection of urease activity revealed that the substitution of Asp29 with Asn29 in UreE resulted in a higher stability of the enzyme complexes. Moreover, the recombinant ES3 showed higher level of urease activity compared to the wildtype without any detectable increase in the expression level of ureC gene, thus highlighting the role of UreE not only in Ni assembly but also on the level of urease activity. During the growth in milk, the recombinant ES3 showed an anticipated urease activity compared to the wildtype, and analogous milk fermentation performance. The overall data obtained by comparing urease-positive and urease-negative strains/mutants confirmed that urease activity strongly impacts on the milk fermentation process and specifically on the yield of the homolactic fermentation.

Intracellular presence of Helicobacter pylori antigen and genes within gastric and vaginal Candida.

BACKGROUND: Helicobacter pylori infections are generally acquired during childhood and affect half of the global population, but its transmission route remains unclear. It is reported that H. pylori can be internalized into Candida, but more evidence is needed for the internalization of H. pylori in human gastrointestinal Candida and vaginal Candida. METHODS: Candida was isolated from vaginal discharge and gastric mucosa biopsies. We PCR-amplified and sequenced H. pylori-specific genes from Candida genomic DNA. Using optical and immunofluorescence microscopy, we identified and observed bacteria-like bodies (BLBs) in Candida isolates and subcultures. Intracellular H. pylori antigen were detected by immunofluorescence using Fluorescein isothiocyanate (FITC)-labeled anti-H. pylori IgG antibodies. Urease activity in H. pylori internalized by Candida was detected by inoculating with urea-based Sabouraud dextrose agar, which changed the agar color from yellow to pink, indicating urease activity. RESULTS: A total of 59 vaginal Candida and two gastric Candida strains were isolated from vaginal discharge and gastric mucosa. Twenty-three isolates were positive for H. pylori 16S rDNA, 12 were positive for cagA and 21 were positive for ureA. The BLBs could be observed in Candida cells, which were positive for H. pylori 16S rDNA, and were viable determined by the LIVE/DEAD BacLight Bacterial Viability kit. Fluorescein isothiocyanate (FITC)-conjugated antibodies could be reacted specifically with H. pylori antigen inside Candida cells by immunofluorescence. Finally, H. pylori-positive Candida remained positive for H. pylori 16S rDNA even after ten subcultures. Urease activity of H. pylori internalized by Candida was positive. CONCLUSION: In the form of BLBs, H. pylori can internalize into gastric Candida and even vaginal Candida, which might have great significance in its transmission and pathogenicity.

New strategies for Helicobacter pylori isolation and sequencing analysis for virulence genes contributing to its pathogenicity.

BACKGROUND: Helicobacter pylori is a fastidious pathogen that is required a complicated medium for growth. Invading epithelial cells of the stomach. H. pylori virulence factors are classified by function, acidic resistivity, adhesion, chemotaxis and motility, molecular mimicry, immunological invasion and modulation, and toxins formation such as cytotoxin-associated genes A (cagA) and vacuolating cytotoxin A (vacA). This study aims to determine a simple and innovative technique to isolate H. pylori from gastric biopsies and assess pathogenicity by virulence factor gene detection. METHODS: A total of 200 patients who were suspected of having H. pylori infection had two antral gastric biopsies undertaken. A rapid urease test (RUT) was used for one, and Brain Heart Infusion broth (BHI) was used to cultivate the other. The molecular study included diagnostics utilizing the 16sRNA housekeeping gene along with the identification of the virulence factors genes (cagA, cagT, and vacA) and sequencing, RESULT: Of the 200 antral gastric biopsies collected, 135 were positive rapid urease tests, and 17 H. pylori isolates were successfully obtained from 135 biopsies. The 16SrRNA as a housekeeping gene is confirmed, and about 53%, 70.5%, and 82.3% of the 17 isolates show carrying cagA, cagT, and vacA genes, respectively. All peptic ulcer isolates have the cagA gene, while Gastroesophageal Reflux Disease (GERD) and non-peptic ulcer disease (NPUD) isolates show the lack of the cagA gene. All bacteria, which were isolated from peptic ulcer, nodular gastritis, and gastritis patients, have a vacA gene. CONCLUSION: The effective method for isolating H. pylori is centrifuging the transport broth after 24 h of incubation. The cagA toxin causes peptic ulcer while vacA toxin induces several histopathological changes in the stomach. Three virulence genes were present in all peptic ulcer-causing bacteria, while only one or none were present in the GERD and NPUD biopsy isolates.

Solid Lipid Nanoparticles Delivering a DNA Vaccine Encoding Helicobacter pylori Urease A Subunit: Immune Analyses before and after a Mouse Model of Infection.

In this study, novel solid lipid particles containing the adjuvant lipid monophosphoryl lipid A (termed 'SLN-A') were synthesised. The SLN-A particles were able to efficiently bind and form complexes with a DNA vaccine encoding the urease alpha subunit of Helicobacter pylori. The resultant nanoparticles were termed lipoplex-A. In a mouse model of H. pylori infection, the lipoplex-A nanoparticles were used to immunise mice, and the resultant immune responses were analysed. It was found that the lipoplex-A vaccine was able to induce high levels of antigen-specific antibodies and an influx of gastric CD4+ T cells in vaccinated mice. In particular, a prime with lipoplex-A and a boost with soluble UreA protein induced significantly high levels of the IgG1 antibody, whereas two doses of lipoplex-A induced high levels of the IgG2c antibody. In this study, lipoplex-A vaccination did not lead to a significant reduction in H. pylori colonisation in a challenge model; however, these results point to the utility of the system for delivering DNA vaccine-encoded antigens to induce immune responses and suggest the ability to tailor those responses.

Reduction of UreB and CagA expression level by siRNA construct in Helicobacter pylori strain SS1.

BACKGROUND: Two important virulence factors, urease and cagA, play an important role in Helicobacter pylori (H. pylori) gastric cancer. Aim of this study was to investigate the expression level and function of ureB and cagA using small interfering RNAs (siRNA). METHODS: SS1 strain of H. pylori was considered as host for natural transformation. siRNA designed for ureB and cagA genes were inserted in pGPU6/GFP/Neo siRNA plasmid vector to evaluate using phenotypic and genotypic approaches. Then, qPCR was performed for determining inhibition rate of ureB and cagA gene expression. RESULTS: The expression levels of siRNA-ureB and siRNA-cagA in the recombinant strain SS1 were reduced by about 5000 and 1000 fold, respectively, compared to the native H. pylori strain SS1. Also, preliminary evaluation of siRNA-ureB in vitro showed inhibition of urea enzyme activity. These data suggest that siRNA may be a powerful new tool for gene silencing in vitro, and for the development of RNAi-based anti-H. pylori therapies. CONCLUSION: Our results show that targeting ureB and cagA genes with siRNA seems to be a new strategy to inhibit urease enzyme activity, reduce inflammation and colonization rate.

Differential expression of urease genes and ureolytic activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa isolates in different nutritional conditions.

Uropathogens have adaptation strategies to survive in the host urinary tract by efficiently utilizing and tolerating the urinary metabolites. Many uropathogens harbour the enzyme urease for the breakdown of urea and the enzymatic breakdown of urea increases the pH and facilitate the struvite crystallization. In this study, the differential urease activity of uropathogenic Escherichia coli and Pseudomonas aeruginosa strains was investigated under different nutritional conditions. The experiments included measurement of growth, pH, urease activity, NH4-N generation and urease gene (ureC) expression among the bacterial strains under different conditions. Further, the implications of urea breakdown on the struvite crystallization in vitro and biofilm formation were also assessed. The study included urease positive isolates and for comparison urease negative isolates were included. Compared to the urease negative strains the urease positive strains formed higher biofilms and motility. The urease positive P. aeruginosa showed significantly higher (p < 0.01) pH and urease activity (A557-A630) compared to E. coli under experimental conditions. Further, supplementation of glucose to the growth media significantly increased the urease activity in P. aeruginosa and in contrast, it was significantly lower in E. coli. The expression profile of urease gene (ureC) was significantly higher (p < 0.001) in P. aeruginosa compared to E. coli and was consistent with the biochemical results of the urease activity under the nutritional conditions. The differential urease activity under two nutritional conditions influenced the biogenic struvite crystallization. It correlated with the urease activity showing higher crystallization rate in P. aeruginosa compared to E. coli. The results highlight the differential urease activity in two common uropathogens under different nutritional conditions that may have significant role on the regulation of virulence, pathogenicity and in the kidney stone disease.

Structural and Functional Analysis of Urease Accessory Protein E from Vancomycin-Resistance Staphylococcus aureus MU50 Strain.

BACKGROUND: An increasing prevalence of biofilm forming strains by vancomycinresistance Staphylococcus aureus (VRSA) is one of the most important causes of antimicrobial resistance. VRSA possesses various regulatory factors to form and sustain biofilm in biotic or abiotic conditions. Among them, ureolytic activity is an important factor in the stabilization of biofilms by neutralizing the acidic environment. Various urease accessory proteins are required to activate the urease enzyme inside the biofilm. OBJECTIVE: To optimize the cloning, expression and purification of urease accessory protein E from VRSA for determination of the secondary structure, and functional characterization by using Berthelot's method. METHODS: BAB58453.1 gene (which encodes possible urease accessory protein E), having 38% similarity to Bacillus pasteurii UreE protein, was cloned, expressed, and purified by single-step affinity chromatography for performing secondary structural studies using circular dichroism spectroscopy, and functional analysis using Berthelot's and crystal violet assay. RESULTS: Structure elucidation using NMR and circular dichroism spectroscopy techniques revealed that UreE protein has a partially foldedα-helical structure. Using Berthelot's method, it was identified that the purified UreE protein has enhanced urease enzyme activity, in comparison to the control. From the results of Berthelot's and crystal violet assays, it was deduced that the selected gene (UreE protein) plays a key role in enhancing urease enzyme activity and contributes to biofilm stability. CONCLUSION: Structural studies on VRSA urease accessory proteins could aid in the identification of new drug targets or the development of effective antibiofilm strategies (in combination with other drug targets) against infections caused by biofilm-producing strains.

Lactiplantibacillus plantarum ZJ316 Reduces Helicobacter pylori Adhesion and Inflammation by Inhibiting the Expression of Adhesin and Urease Genes.

SCOPE: The present study aims to investigate the anti-Helicobacter pylori (H. pylori) effects of Lactiplantibacillus plantarum ZJ316 (L. plantarum ZJ316) both in vitro and in vivo. METHODS AND RESULTS: This study finds that L. plantarum ZJ316 effectively suppresses H. pylori adhesion in inhibition (Pre-ZJ316), competition (Co-ZJ316), and displacement (Post-ZJ316) assays, and Pre-ZJ316 displaying the most potent inhibitory effect with an impressive inhibition ratio of 70.14%. Upon anti-adhesion, L. plantarum ZJ316 significantly downregulates the expression of H. pylori virulence genes, including ureA, ureB, flaA, and sabA, with inhibition ratios of 46.83%, 24.02%, 21.42%, and 62.38% at 2 h, respectively. In addition, L. plantarum ZJ316 is observed to reduce the level of interleukin 8 (IL-8) and improve cell viability in infected AGS cells. Furthermore, in vivo studies show that supplementation with L. plantarum ZJ316 effectively hinders H. pylori colonization and significantly suppresses the infiltration of immune cells and IL-8 production with H. pylori infection, protecting host from inflammatory damage. CONCLUSION: L. plantarum ZJ316 exhibits excellent adhesion inhibition on H. pylori, and may be used as a probiotic candidate in the prevention or adjuvant therapy of gastric disease caused by H. pylori.

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines.

BACKGROUND: Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR. MATERIALS AND METHODS: We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori. RESULTS: Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log. CONCLUSIONS: This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.

[Research progress of Helicobacter pylori vaccine].

Helicobacter pylori (Hp) is one of most common pathogens causing gastrointestinal disorder including gastric ulcer, duodenal ulcer and gastric cancer, etc. It has been verified as class I carcinogen by WHO. Nowadays, combination antibiotics and proton pump inhibitor are mainly used to erase Hp in clinical application. However, with the increased resistance of Hp, the vaccine against Hp might become the best strategy to eradicate Hp. Elements including urease, virulence factor, outer membrane protein, flagella, play an important role in Hp infection, colonization and reproduction. They have become potential candidate antigens in the development of Hp vaccine, as reported in previous studies. Presently, these antigens-centric vaccines have been tested in animal models. Therefore, this article reviews the studies on Hp vaccine with urease, virulence genes, outer membrane protein and flagella as their candidate antigens, in an attempt to provide insights for research in this regard.

Collinsella urealyticum sp. nov., a urease-positive bacterial strain isolated from swine faeces.

A novel actinobacterial strain, designated AGMB00827T, was isolated from swine faeces. Strain AGMB00827T was obligately anaerobic, Gram-stain-positive, non-motile, non-spore-forming and rod-shaped bacterium. Comparative analyses based on the 16S rRNA gene and whole genome sequence revealed that strain AGMB00827T was affiliated to the genus Collinsella, and was most closely related to Collinsella vaginalis Marseille-P2666T (= KCTC 25056T). Biochemical analysis showed strain AGMB00827T was negative for catalase and oxidase. Interestingly, strain AGMB00827T possessed urease activity, which was determined by traditional methods (API test and Christensen's urea medium), unlike related strains. Furthermore, the major cellular fatty acids (> 10%) of the isolate were C18:1 ω9c, C16:0, C16:0 DMA and C18:2 ω9,12c DMA. Based on the whole genome sequence analysis, the DNA G + C content of strain AGMB00827T was 52.3%, and the genome size and numbers of rRNA and tRNA genes were 1,945,251 bp, 3 and 46, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain AGMB00827T and C. vaginalis KCTC 25056 T were 71.0 and 23.2%, respectively. Additionally, the genome analysis revealed that strain AGMB00827T possesses urease gene cluster including ureABC and ureDEFG while the related strains do not have those genes, which is consistent with the urease activity. On the basis of polyphasic taxonomic approach, strain AGMB00827T represents a novel species within the genus Collinsella, for which the name Collinsella urealyticum sp. nov. is proposed. The type strain is AGMB00827T (= KCTC 25287T = GDMCC 1.2724T).

Functional gene-guided enrichment plus in situ microsphere cultivation enables isolation of new crucial ureolytic bacteria from the rumen of cattle.

BACKGROUND: Ruminants can utilize urea as a dietary nitrogen source owing to their ability to recycle urea-N back to the rumen where numerous ureolytic bacteria hydrolyze urea into ammonia, which is used by numerous bacteria as their nitrogen source. Rumen ureolytic bacteria are the key microbes making ruminants the only type of animals independent of pre-formed amino acids for survival, thus having attracted much research interest. Sequencing-based studies have helped gain new insights into ruminal ureolytic bacterial diversity, but only a limited number of ureolytic bacteria have been isolated into pure cultures or studied, hindering the understanding of ureolytic bacteria with respect to their metabolism, physiology, and ecology, all of which are required to effectively improve urea-N utilization efficiency. RESULTS: We established and used an integrated approach, which include urease gene (ureC) guided enrichment plus in situ agarose microsphere embedding and cultivation under rumen-simulating conditions, to isolate ureolytic bacteria from the rumen microbiome. We optimized the dilutions of the rumen microbiome during the enrichment, single-cell embedding, and then in situ cultivation of microsphere-embedded bacteria using dialysis bags placed in rumen fluid. Metabonomic analysis revealed that the dialysis bags had a fermentation profile very similar to the simulated rumen fermentation. In total, we isolated 404 unique strains of bacteria, of which 52 strains were selected for genomic sequencing. Genomic analyses revealed that 28 strains, which were classified into 12 species, contained urease genes. All these ureolytic bacteria represent new species ever identified in the rumen and represented the most abundant ureolytic species. Compared to all the previously isolated ruminal ureolytic species combined, the newly isolated ureolytic bacteria increased the number of genotypically and phenotypically characterized ureolytic species by 34.38% and 45.83%, respectively. These isolated strains have unique genes compared to the known ureolytic strains of the same species indicating their new metabolic functions, especially in energy and nitrogen metabolism. All the ureolytic species were ubiquitous in the rumen of six different species of ruminants and were correlated to dietary urea metabolism in the rumen and milk protein production. We discovered five different organizations of urease gene clusters among the new isolates, and they had varied approaches to hydrolyze urea. The key amino acid residues of the UreC protein that potentially plays critical regulatory roles in urease activation were also identified. CONCLUSIONS: We established an integrated methodology for the efficient isolation of ureolytic bacteria, which expanded the biological resource of crucial ureolytic bacteria from the rumen. These isolates play a vital role in the incorporation of dietary nitrogen into bacterial biomass and hence contribute to ruminant growth and productivity. Moreover, this methodology can enable efficient isolation and cultivation of other bacteria of interest in the environment and help bridge the knowledge gap between genotypes and phenotypes of uncultured bacteria. Video abstract.

Regulation of Helicobacter pylori Urease and Acetone Carboxylase Genes by Nitric Oxide and the CrdRS Two-Component System.

Helicobacter pylori colonizes the human gastric mucosa and causes various gastroduodenal diseases, including peptic ulceration and gastric cancer. Colonization requires the actions of two-component systems (TCSs) to sense and respond to changes in the host environment. In this study, we evaluated gene regulation mediated by the CrdRS TCS. Few studies have evaluated this TCS, leaving the signal(s) yet to be exhaustively determined and a need for a more complete regulon to be delineated. We performed RNA sequencing (RNA-Seq) on three isogenic H. pylori 26695 mutants: a control, a mutant with deletion of the sensory histidine kinase, ΔcrdS, and a mutant with deletion of the response regulator, ΔcrdR. Comparison of the RNA-Seq results from these mutants established a 40-gene regulon putatively controlled by the CrdRS TCS. Quantitative reverse transcriptase PCR (RT-qPCR) was used to validate 7 of 11 putative regulon members selected for analysis. We further investigated 6 confirmed CrdRS regulon genes by using phospho-incompetent H. pylori 26695 CrdR D53A and CrdS H173A mutants. These experiments further confirmed the role of CrdRS in regulation of urease, acetone carboxylase, hofD, and HP1440. Expression of these CrdRS regulon genes was also evaluated under 10 µM nitric oxide (NO) conditions. This revealed that ureA, acxA, hofD, and HP1440 expression is affected by NO in a CrdRS-dependent manner. Importantly, three of these genes (ureA, acxA, and hofD) are known to play important roles in H. pylori colonization of the stomach. IMPORTANCE The molecular strategies used by Helicobacter pylori to colonize and persist in the harsh environment of the human stomach are a critical area of study. Our study identified several genes in this gastric pathogen, including ureA, a gene encoding a protein essential to the survival of H. pylori, that are regulated via the CrdRS two-component system (TCS) in response to nitric oxide (NO). NO is a product of the innate immune system of the human host. The identification of these genes whose expression is regulated by this molecule may give insights to novel therapeutics. Two genes (ureA and acxA) determined in this study to be regulated by NO via CrdRS have been previously determined to be regulated by other TCSs, indicating that the expression of these genes may be of critical importance to H. pylori.

The Ability of Streptococcus thermophilus BT01 to Modulate Urease Activity in Healthy Subjects' Fecal Samples Depends on the Biomass Production Process.

SCOPE: This study evaluates how manufacturing conditions of probiotic biomass production, using two different cryoprotectants, Cryo-A and Cryo-B, can affect Streptococcus thermophilus BT01 in vivo gastrointestinal tract survival and its ability to modulate the level of urease activity in fecal samples of healthy subjects. METHODS AND RESULTS: A randomized controlled cross-over study is carried out on 20 adult healthy subjects to evaluate total and viable loads, persistence of S. thermophilus BT01, and urease activity in fecal samples. Strain-specific quantification by using developed culture-based method and molecular qPCR tool allows to quantify viable S. thermophilus BT01 strain in 90% of the subjects. The quantification of both total DNA and recovered viable S. thermophilus BT01 in fecal samples does not reveal significant differences between Cryo-A or Cryo-B treated biomass. However, the administration of S. thermophilus BT01 produced with Cryo-A results in a decreased urease activity in fecal samples compared to Cryo-B protected cells. CONCLUSION: This study i) highlights how the manufacturing conditions can play a role in influencing the probiotic functionality in vivo and ii) represents the first evidence that links S. thermophilus to a specific probiotic mechanism, the reduction of urease activity in fecal samples.

Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana.

CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.

Research progress of Helicobacter pylori vaccine / 细胞与分子免疫学杂志

Helicobacter pylori (Hp) is one of most common pathogens causing gastrointestinal disorder including gastric ulcer, duodenal ulcer and gastric cancer, etc. It has been verified as class I carcinogen by WHO. Nowadays, combination antibiotics and proton pump inhibitor are mainly used to erase Hp in clinical application. However, with the increased resistance of Hp, the vaccine against Hp might become the best strategy to eradicate Hp. Elements including urease, virulence factor, outer membrane protein, flagella, play an important role in Hp infection, colonization and reproduction. They have become potential candidate antigens in the development of Hp vaccine, as reported in previous studies. Presently, these antigens-centric vaccines have been tested in animal models. Therefore, this article reviews the studies on Hp vaccine with urease, virulence genes, outer membrane protein and flagella as their candidate antigens, in an attempt to provide insights for research in this regard.

Multiple regulatory mechanisms for pH homeostasis in the gastric pathogen, Helicobacter pylori.

Acid-resistance in gastric pathogen Helicobacter pylori requires the coordination of four essential processes to regulate urease activity. Firstly, urease expression above a base level needs to be finely tuned at different ambient pH. Secondly, as nickel is needed to activate urease, nickel homeostasis needs to be maintained by proteins that import and export nickel ions, and sequester, store and release nickel when needed. Thirdly, urease accessary proteins that activate urease activity by nickel insertion need to be expressed. Finally, a reliable source of urea needs to be maintained by both intrinsic and extrinsic sources of urea. Two-component systems (arsRS and flgRS), as well as a nickel response regulator (NikR), sense the change in pH and act on a variety of genes to accomplish the function of acid resistance without causing cellular overalkalization and nickel toxicity. Nickel storage proteins also feature built-in switches to store nickel at neutral pH and release nickel at low pH. This review summarizes the current status of H. pylori research and highlights a number of hypotheses that need to be tested.

Cellulomonas dongxiuzhuiae sp. nov., Cellulomonas wangleii sp. nov. and Cellulomonas fengjieae sp. nov., isolated from the intestinal contents of Marmota himalayana.

Six Gram-stain-positive, aerobic or facultative anaerobic, catalase-positive, urease- and oxidase-negative, rod-shaped bacteria (zg-ZUI157T/zg-ZUI40, zg-ZUI222T/zg-ZUI199 and zg-ZUI188T/ zg-ZUI168) were characterized by a polyphasic approach. Optimal growth of the six strains was observed at pH 7.0 and 28 °C. Phylogenetic analyses based on the 16S rRNA gene and 247 core genes revealed that they belong to genus Cellulomonas. The three type strains have low digital DNA-DNA hybridization (19.3-30.1%) and average nucleotide identity values (78.0-85.5%) with all available genomes in the genus Cellulomonas, and a DNA G+C content range of 73.0-74.6 mol%. The major fatty acids detected in strain pairs zg-ZUI157T/zg-ZUI40 and zg-ZUI 222T/zg-ZUI199 were C16:0, anteiso-C15:0 and anteiso A-C15:1, and C16:0, anteiso-C15:0, anteiso A-C15:1 and anteiso-C17:0 in strain pair zg-ZUI188T/zg-ZUI168. Diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol mannosides were the major polar lipids detected in the three novel species. MK-9(H4) was the predominant quinone detected in strains zg-ZUI222T (87.4 %) and zg-ZUI188T (91.4 %), and MK-9(H4) (49.1 %) and MK-8 (43.4 %) in strain zg-ZUI157T. The cell-wall sugars detected in the three novel species mainly contained rhamnose. The cell-wall peptidoglycan type of the three novel species was A4ß, with an inferred l-Orn-d-Asp interpeptide bridge for strains zg-ZUI157T and zg-ZUI222T, and l-Orn-d-Glu for strain zg-ZUI188T. Based on the results of the phenotypic, phylogenetic, genomic hybridization, average nucleotide identity and chemotaxonomic analyses, the six strains should be classified as belonging to three novel Cellulomonas species, for which the names Cellulomonas dongxiuzhuiae sp. nov. (zg-ZUI157T=GDMCC 1.2559T=KCTC 49678T), Cellulomonas wangleii sp. nov. (zg-ZUI222T=GDMCC 1.2501T=KCTC 49675T) and Cellulomonas fengjieae sp. nov. (zg-ZUI188T=GDMCC 1.2563T=KCTC 49674T) are proposed.

Streptomyces solincola sp. nov., isolated from soil in Malaysia.

A urease-producing Gram-stain-positive actinobacterium, designated strain T5T, was isolated from a soil sample collected at a highway hillslope in Selangor, Malaysia. The strain was found to produce pale yellowish-pink aerial mycelia with smooth long chain spores and extensively branched light yellowish-pink substrate mycelia on oatmeal agar. Strain T5T grew at 15-37 °C, pH 6-11, and tolerated up to 9 % (w/v) NaCl, with optimal growth occurring at 28 °C, pH 6-9 and without NaCl. The whole-cell sugar hydrolysate of strain T5T contained galactose, glucose and ribose. The ll-diaminopimelic acid isomer was detected in the cell wall. Diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were found to be the predominant polar lipids. The main fatty acids were anteiso-C17 : 0, iso-C16 : 0, anteiso-C15 : 0 and iso-C14 : 0. Comparative analysis of the 16S rRNA gene sequences indicated that strain T5T belonged to Streptomyces of the family Streptomycetaceae with the highest 16S rRNA gene sequence similarity to Streptomyces lichenis LCR6-01T (99.0 %). The overall genome relatedness indices revealed that the closest related species was S. lichenis LCR6-01T with 89.4 % average nucleotide identity and 33.7 % digital DNA-DNA hybridization. Phylogeny analyses showed that strain T5T was closely related to Streptomyces fradiae, Streptomyces lavendofoliae, Streptomyces lichenis, Streptomyces roseolilacinus and Streptomyces somaliensis. Based on these polyphasic data, strain T5T represents a novel species, for which the name Streptomyces solincola sp. nov. is proposed. The type strain is T5T (=TBRC 5137T= DSM 42166T).