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.

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.

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.

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).

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.

The pathogenic characterization of Citrobacter freundii and its activation on immune related genes in Macrobrachium nipponense.

Out breaks of mass mortalities occurred in Macrobrachium nipponense farms in Jintan county, Jiangsu Province. The bacterial isolates from M. nipponense exhibited the same phenotypic traits and biochemical characteristics, and were identified as Citrobacter freundii according to biochemical characteristics and molecular identification. The infection test revealed that the strain YG2 was pathogenic to M. nipponense, and the half lethal dose (LD50) was 3.35 × 105 CFU/mL at 7 d post-infection. Detection of virulence genes indicated that YG2 was positive for cfa, ureG, ureF, ureE, ureD, viaB, ompX, and LDH. Furthermore, the results of extracellular enzyme analysis revealed that the strain can produce protease, amylase, lecithin, urease, and hemolysin. Antibiotic resistance results showed that the isolate was resistant to ampicillin, cefazolin, cephalothin, cefoxitin, aboren, doxycycline, neomycin, penicillin, erythromycin, and vancomycin. The expression level of MyD88, α2M, CDSP, and Relish were detected in hepatopancreas, hemolymph, gills and intestine tissues by quantitive real-time PCR (qRT-PCR), and clear transcriptional activation of these genes were observed in M. nipponense after C. freundii infection. These results revealed pathogenicity of C. freundii and its activation of host immune response, which will provide a scientific reference for the breeding and disease prevention in M. nipponense culture.

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).

Expression, Purification, and Comparative Inhibition of Helicobacter pylori Urease by Regio-Selectively Alkylated Benzimidazole 2-Thione Derivatives.

The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol-1; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.

Ynt is the primary nickel import system used by Proteus mirabilis and specifically contributes to fitness by supplying nickel for urease activity.

Proteus mirabilis is a Gram-negative uropathogen and frequent cause of catheter-associated urinary tract infection (CAUTI). One important virulence factor is its urease enzyme, which requires nickel to be catalytically active. It is, therefore, hypothesized that nickel import is critical for P. mirabilis urease activity and pathogenesis during infection. P. mirabilis strain HI4320 encodes two putative nickel import systems, designated Nik and Ynt. By disrupting the substrate-binding proteins from each import system (nikA and yntA), we show that Ynt is the primary nickel importer, while Nik only compensates for loss of Ynt at high nickel concentrations. We further demonstrate that these are the only binding proteins capable of importing nickel for incorporation into the urease enzyme. Loss of either nickel-binding protein results in a significant fitness defect in a murine model of CAUTI, but YntA is more crucial as the yntA mutant was significantly outcompeted by the nikA mutant. Furthermore, despite the importance of nickel transport for hydrogenase activity, the sole contribution of yntA and nikA to virulence is due to their role in urease activity, as neither mutant exhibited a fitness defect when disrupted in a urease-negative background.

Microscale heterogeneity of the soil nitrogen cycling microbial functional structure and potential metabolism.

Soil aggregates, with complex spatial and nutritional heterogeneity, are clearly important for regulating microbial community ecology and biogeochemistry in soils. However, how the taxonomic composition and functional attributes of N-cycling-microbes within different soil particle-size fractions under a long-term fertilization treatment remains largely unknown. Here, we examined the composition and metabolic potential for urease activity, nitrification, N2 O production and reduction of the microbial communities attached to different sized soil particles (2000-250, 250-53 and <53 µm) using a functional gene microarray (GeoChip) and functional assays. We found that urease activity and nitrification were higher in <53 µm fractions, whereas N2 O production and reduction rates were greater in 2000-250 and 250-53 µm across different fertilizer regimes. The abundance of key N-cycling genes involved in anammox, ammonification, assimilatory and dissimilatory N reduction, denitrification, nitrification and N2 -fixation detected by GeoChip increased as soil aggregate size decreased; and the particular key genes abundance (e.g., ureC, amoA, narG, nirS/K) and their corresponding activity were uncoupled. Aggregate fraction exerted significant impacts on N-cycling microbial taxonomic composition, which was significantly shaped by soil nutrition. Taken together, these findings indicate the important roles of soil aggregates in differentiating N-cycling metabolic potential and taxonomic composition, and provide empirical evidence that nitrogen metabolism potential and community are uncoupled due to aggregate heterogeneity.

Urease is an essential component of the acid response network of Staphylococcus aureus and is required for a persistent murine kidney infection.

Staphylococcus aureus causes acute and chronic infections resulting in significant morbidity. Urease, an enzyme that generates NH3 and CO2 from urea, is key to pH homeostasis in bacterial pathogens under acidic stress and nitrogen limitation. However, the function of urease in S. aureus niche colonization and nitrogen metabolism has not been extensively studied. We discovered that urease is essential for pH homeostasis and viability in urea-rich environments under weak acid stress. The regulation of urease transcription by CcpA, Agr, and CodY was identified in this study, implying a complex network that controls urease expression in response to changes in metabolic flux. In addition, it was determined that the endogenous urea derived from arginine is not a significant contributor to the intracellular nitrogen pool in non-acidic conditions. Furthermore, we found that during a murine chronic renal infection, urease facilitates S. aureus persistence by promoting bacterial fitness in the low-pH, urea-rich kidney. Overall, our study establishes that urease in S. aureus is not only a primary component of the acid response network but also an important factor required for persistent murine renal infections.

Synthesis, X-ray, Hirshfeld surface analysis, exploration of DNA binding, urease enzyme inhibition and anticancer activities of novel adamantane-naphthyl thiourea conjugate.

1-(adamantane-1-carbonyl-3-(1-naphthyl)) thiourea (C22H24N2OS (4), was synthesized by the reaction of freshly prepared adamantane-1-carbonyl chloride from corresponding acid (3) with ammonium thiocyanate in 1:1 M ratio in dry acetone to afford the adamantane-1-carbonyl isothiocyanate (2) in situ followed by treatment with 1-naphthyl amine (3). The structure was established by elemental analyses, FTIR, 1H, 13C NMR and mass spectroscopy. The molecular and crystal structure were determined by single crystal X-ray analysis. It belongs to triclinic system P - 1 space group with a = 6.7832(5) Å, b = 11.1810(8) Å, c = 13.6660(10) Å, α = 105.941(6)°, ß = 103.730(6)°, γ = 104.562(6)°, Z = 2, V = 910.82(11) Å3. The naphthyl group is almost planar. In the crystal structure, intermolecular CH···O hydrogen bonds link the molecules into centrosymmetric dimers, enclosing R22(14) ring motifs, while the intramolecular NH···O hydrogen bonds enclose S(6) ring motifs, in which they may be effective in the stabilization of the structure. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H … H (59.3%), H … C/C … H (19.8%) and H … S/S … H (10.1%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing. DFT, molecular docking and urease inhibition studies revealed stability and electron withdrawing nature of 4 as compared to DNA base pairs and residues of urease. The DNA binding results from docking, UV- visible spectroscopy, and viscosity studies indicated significant binding of 4 with the DNA via intercalation and groove binding. Further investigation of the compound was done on hepatocellular carcinoma; Huh-7 cell line as well as normal human embryonic kidney; Hek-293 cell line. The compound showed significant cytotoxic activity against Huh-7 cells in comparison to normal Hek-293 cells indicating selective cytotoxicity towards cancer cells.

Acid-responsive activity of the Helicobacter pylori metalloregulator NikR.

Helicobacter pylori is a human pathogen that infects the stomach, where it experiences variable pH. To survive the acidic gastric conditions, H. pylori produces large quantities of urease, a nickel enzyme that hydrolyzes urea to ammonia, which neutralizes the local environment. One of the regulators of urease expression in H. pylori is HpNikR, a nickel-responsive transcription factor. Here we show that HpNikR also regulates urease expression in response to changes in pH, linking acid adaptation and nickel homeostasis. Upon measuring the cytosolic pH of H. pylori exposed to an external pH of 2, similar to the acidic shock conditions that occur in the human stomach, a significant drop in internal pH was observed. This decrease in internal pH resulted in HpNikR-dependent activation of ureA transcription. Furthermore, analysis of a slate of H. pylori genes encoding other acid adaptation or nickel homeostasis components revealed HpNikR-dependent regulation in response to acid shock. This regulation was consistent with pH-dependent DNA binding to the corresponding promoter sequences observed in vitro with purified HpNikR. These results demonstrate that HpNikR can directly respond to changes in cytosolic pH during acid acclimation and illustrate the exquisitely coordinated regulatory networks that support H. pylori infections in the harsh environment of the human stomach.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

The virulence factor urease and its unexplored role in the metabolism of Cryptococcus neoformans.

Cryptococcal urease is believed to be important for the degradation of exogenous urea that the yeast encounters both in its natural environment and within the human host. Endogenous urea produced by the yeast's own metabolic reactions, however, may also serve as a substrate for the urease enzyme. Using wild-type, urease-deletion mutant and urease-reconstituted strains of Cryptococcus neoformans H99, we studied reactions located up- and downstream from endogenous urea. We demonstrated that urease is important for cryptococcal growth and that, compared to nutrient-rich conditions at 26°C, urease activity is higher under nutrient-limited conditions at 37°C. Compared to cells with a functional urease enzyme, urease-deficient cells had significantly higher intracellular urea levels and also showed more arginase activity, which may act as a potential source of endogenous urea. Metabolic reactions linked to arginase were also affected, since urease-positive and urease-negative cells differed with respect to agmatinase activity, polyamine synthesis, and intracellular levels of proline and reactive oxygen species. Lastly, urease-deficient cells showed higher melanin levels at 26°C than wild-type cells, while the inverse was observed at 37°C. These results suggest that cryptococcal urease is associated with the functioning of key metabolic pathways within the yeast cell.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii.

BACKGROUND: The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. RESULTS: The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. CONCLUSIONS: Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

Unraveling the factors and mechanism involved in persistence: Host-pathogen interactions in Helicobacter pylori.

Helicobacter pylori and humans have one of the most complex relationships in nature. How a bacterium manages to live in one of the harshest and hostile environments is a topic of unraveling mysteries. H. pylori is a prevalent species and it colonizes the human gut of more than 50% of the world population. It infects the epithelial region of antrum and persists there for a long period. Over the time of evolution, H. pylori has developed complex strategies to extend the degree of inflammation in gastric mucosa. H. pylori needs specific adaptations for initial colonization into the host environment like helical shape, flagellar movement, chemotaxis, and the production of urease enzyme that neutralizes acidic environment of the stomach. There are several factors from the bacterium as well as from the host that participate in these complex interactions. On the other hand, to establish the persistent infection, H. pylori escapes the immune system by mimicking the host antigens. This pathogen has the ability to dodge the immune system and then persist there in the form of host cell, which leads to immune tolerance. H. pylori has an ability to manipulate its own pathogen-associated molecular patterns, which leads to an inhibition in the binding with specific pattern recognition receptors of the host to avoid immune cell detection. Also, it manipulates the host metabolic homeostasis in the gastric epithelium. Besides, it has several genes, which may get involved in the acquisition of nutrition from the host to survive longer in the host. Due to the persistence of H. pylori, it causes chronic inflammation and raises the chances of gastric cancer. This review highlights the important elements, which are certainly responsible for the persistence of H. pylori in the human host.