A remarkable peroxidase-like behavior of the catalase KatA from the pathogenic bacteria Helicobacter pylori: The oxidation reaction with formate as substrate and the stabilization of an [Fe(IV) = O Trp•] intermediate assessed by multifrequency EPR spectroscopy.

We have characterized the catalytic cycle of the Helicobacter pylori KatA catalase (HPC). H. pylori is a human and animal pathogen responsible for gastrointestinal infections. Multifrequency (9-285 GHz) EPR spectroscopy was applied to identify the high-valent intermediates (5 ≤ pH ≤ 8.5). The broad (2000 G) 9-GHz EPR spectrum consistent with the [Fe(IV) = O Por•+] intermediate was detected, and showed a clear pH dependence on the exchange-coupling of the radical (delocalized over the porphyrin moiety) due to the magnetic interaction with the ferryl iron. In addition, Trp• (for pH ≤ 6) and Tyr• (for 5 ≤ pH ≤ 8.5) species were distinguished by the advantageous resolution of their g-values in the 285-GHz EPR spectrum. The unequivocal identification of the high-valent intermediates in HPC by their distinct EPR spectra allowed us to address their reactivity towards substrates. The stabilization of an [Fe(IV) = O Trp•] species in HPC, unprecedented in monofunctional catalases and possibly involved in the oxidation of formate to the formyloxyl radical at pH ≤ 6, is reminiscent of intermediates previously identified in the catalytic cycle of bifunctional catalase-peroxidases. The 2e- oxidation of formate by the [Fe(IV) = O Por•+] species, both at basic and acidic pH conditions, involving a 1H+/2e- oxidation in a cytochrome P450 peroxygenase-like reaction is proposed. Our findings demonstrate that moonlighting by the H. pylori catalase includes formate oxidation, an enzymatic reaction possibly related to the unique strategy of the neutrophile bacterium for gastric colonization, that is the release of CO2 to regulate the pH in the acidic environment.

Machine Learning-Driven Classification of Urease Inhibitors Leveraging Physicochemical Properties as Effective Filter Criteria.

Urease, a pivotal enzyme in nitrogen metabolism, plays a crucial role in various microorganisms, including the pathogenic Helicobacter pylori. Inhibiting urease activity offers a promising approach to combating infections and associated ailments, such as chronic kidney diseases and gastric cancer. However, identifying potent urease inhibitors remains challenging due to resistance issues that hinder traditional approaches. Recently, machine learning (ML)-based models have demonstrated the ability to predict the bioactivity of molecules rapidly and effectively. In this study, we present ML models designed to predict urease inhibitors by leveraging essential physicochemical properties. The methodological approach involved constructing a dataset of urease inhibitors through an extensive literature search. Subsequently, these inhibitors were characterized based on physicochemical properties calculations. An exploratory data analysis was then conducted to identify and analyze critical features. Ultimately, 252 classification models were trained, utilizing a combination of seven ML algorithms, three attribute selection methods, and six different strategies for categorizing inhibitory activity. The investigation unveiled discernible trends distinguishing urease inhibitors from non-inhibitors. This differentiation enabled the identification of essential features that are crucial for precise classification. Through a comprehensive comparison of ML algorithms, tree-based methods like random forest, decision tree, and XGBoost exhibited superior performance. Additionally, incorporating the "chemical family type" attribute significantly enhanced model accuracy. Strategies involving a gray-zone categorization demonstrated marked improvements in predictive precision. This research underscores the transformative potential of ML in predicting urease inhibitors. The meticulous methodology outlined herein offers actionable insights for developing robust predictive models within biochemical systems.

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

Synthesis and Biological Evaluation of Dithiobisacetamides as Novel Urease Inhibitors.

Thirty-eight disulfides containing N-arylacetamide were designed and synthesized in an effort to develop novel urease inhibitors. Biological evaluation revealed that some of the synthetic compounds exhibited strong inhibitory potency against both cell-free urease and urease in intact cell with low cytotoxicity to mammalian cells even at concentration up to 250 µM. Of note, 2,2'-dithiobis(N-(2-fluorophenyl)acetamide) (d7), 2,2'-dithiobis(N-(3,5-difluorophenyl)acetamide) (d24), and 2,2'-dithiobis(N-(3-fluorophenyl)acetamide) (d8) were here identified as the most active inhibitors with IC50 of 0.074, 0.44, and 0.81 µM, showing 32- to 355-fold higher potency than the positive control acetohydroxamic acid. These disulfides were confirmed to bind urease without covalent modification of the cysteine residue and to inhibit urease reversibly with a mixed inhibition mechanism. They also showed very good anti-Helicobacter pylori activities with d8 showing a comparable potency to the clinical used drug amoxicillin. The impressive in vitro biological profile indicated their immense potential as therapeutic agents to tackle H. pylori caused infections.

MdaB and NfrA, Two Novel Reductases Important in the Survival and Persistence of the Major Enteropathogen Campylobacter jejuni.

The paralogues RrpA and RrpB, which are members of the MarR family of DNA binding proteins, are important for the survival of the global bacterial foodborne pathogen Campylobacter jejuni under redox stress. We report that RrpA is a positive regulator of mdaB, encoding a flavin-dependent quinone reductase that contributes to the protection from redox stress mediated by structurally diverse quinones, while RrpB negatively regulates the expression of cj1555c (renamed nfrA for NADPH-flavin reductase A), encoding a flavin reductase. NfrA reduces riboflavin at a greater rate than its derivatives, suggesting that exogenous free flavins are the natural substrate. MdaB and NfrA both prefer NADPH as an electron donor. Cysteine substitution and posttranslational modification analyses indicated that RrpA and RrpB employ a cysteine-based redox switch. Complete genome sequence analyses revealed that mdaB is frequently found in Campylobacter and related Helicobacter spp., while nfrA is predominant in C. jejuni strains. Quinones and flavins are redox cycling agents secreted by a wide range of cell types that can form damaging superoxide by one-electron reactions. We propose a model for stress adaptation where MdaB and NfrA facilitate a two-electron reduction mechanism to the less toxic hydroquinones, thus aiding survival and persistence of this major pathogen. IMPORTANCE Changes in cellular redox potential result in alteration in the oxidation state of intracellular metabolites and enzymes; consequently, cells make adjustments that favor growth and survival. The work we present here answers some of the many questions that have remained elusive over the years of investigation into the enigmatic microaerophile bacterium Campylobacter jejuni. We employed molecular approaches to understand the regulation mechanisms and functional analyses to reveal the roles of two novel quinone and flavin reductases; both serve as major pools of cellular redox-active molecules. This work extends our knowledge on bacterial redox sensing mechanisms and the significance of hemostasis.

Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase.

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.

The Helicobacter pylori CagY Protein Drives Gastric Th1 and Th17 Inflammation and B Cell Proliferation in Gastric MALT Lymphoma.

BACKGROUND: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. METHODS: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori-infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. RESULTS: 22 of 158 CD4+ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4+ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4+ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. CONCLUSION: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.

Identification and In Silico Characterization of Novel Helicobacter pylori Glucose-6-Phosphate Dehydrogenase Inhibitors.

Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Helicobacter pylori PqqE is a new virulence factor that cleaves junctional adhesion molecule A and disrupts gastric epithelial integrity.

Helicobacter pylori infects approximately half of the world's population and is the strongest risk factor for peptic ulcer disease and gastric cancer, representing a major global health concern. H. pylori persistently colonizes the gastric epithelium, where it subverts the highly organized structures that maintain epithelial integrity. Here, a unique strategy used by H. pylori to disrupt the gastric epithelial junctional adhesion molecule-A (JAM-A) is disclosed, using various experimental models that include gastric cell lines, primary human gastric cells, and biopsy specimens of infected and non-infected individuals. H. pylori preferentially cleaves the cytoplasmic domain of JAM-A at Alanine 285. Cells stably transfected with full-length JAM-A or JAM-A lacking the cleaved sequence are used in a range of functional assays, which demonstrate that the H. pylori cleaved region is critical to the maintenance of the epithelial barrier and of cell-cell adhesion. Notably, by combining chromatography techniques and mass spectrometry, PqqE (HP1012) is purified and identified as the H. pylori virulence factor that cleaves JAM-A, uncovering a previously unreported function for this bacterial protease. These findings propose a novel mechanism for H. pylori to disrupt epithelial integrity and functions, breaking new ground in the understanding of the pathogenesis of this highly prevalent and clinically relevant infection.

Helicobacter pylori Xanthine-Guanine-Hypoxanthine Phosphoribosyltransferase-A Putative Target for Drug Discovery against Gastrointestinal Tract Infections.

Helicobacter pylori (Hp) is a human pathogen that lives in the gastric mucosa of approximately 50% of the world's population causing gastritis, peptic ulcers, and gastric cancer. An increase in resistance to current drugs has sparked the search for new Hp drug targets and therapeutics. One target is the disruption of nucleic acid production, which can be achieved by impeding the synthesis of 6-oxopurine nucleoside monophosphates, the precursors of DNA and RNA. These metabolites are synthesized by Hp xanthine-guanine-hypoxanthine phosphoribosyltransferase (XGHPRT). Here, nucleoside phosphonates have been evaluated, which inhibit the activity of this enzyme with Ki values as low as 200 nM. The prodrugs of these compounds arrest the growth of Hp at a concentration of 50 µM in cell-based assays. The kinetic properties of HpXGHPRT have been determined together with its X-ray crystal structure in the absence and presence of 9-[(N-3-phosphonopropyl)-aminomethyl-9-deazahypoxanthine, providing a basis for new antibiotic development.

Endoscopic Detection of Helicobacter pylori by the Rapid Urease Test.

Helicobacter pylori infection can be detected on endoscopic biopsy of the gastric mucosa, by means of several techniques. The biopsy specimens are usually taken from the prepyloric region, but additional biopsy specimens obtained proximally increase the sensitivity of invasive tests and are recommended, especially if the patient has recently been treated with a proton-pump inhibitor. The effects of an increased risk of sampling error and the lower prevalence of H. pylori infection on the diagnostic accuracy of standard invasive tests needs to be considered. Despite evidence of enhanced yield with additional biopsies, combined Rapid Urease Tests (RUTs) have not been widely adopted. The other endoscopic tests, histology , and culture are also prone to sampling error and adoption of appropriate biopsy protocols should be widely adopted to enhance diagnostic yield.

A comprehensive review on the role of protein tyrosine phosphatases in gastric cancer development and progression.

The main post-translational reversible modulation of proteins is phosphorylation and dephosphorylation, catalyzed by protein kinases (PKs) and protein phosphatases (PPs) which is crucial for homeostasis. Imbalance in this crosstalk can be related to diseases, including cancer. Plenty of evidence indicates that protein tyrosine phosphatases (PTPs) can act as tumor suppressors and tumor promoters. In gastric cancer (GC), there is a lack of understanding of the molecular aspects behind the tumoral onset and progression. Here we describe several members of the PTP family related to gastric carcinogenesis. We discuss the associated molecular mechanisms which support the down or up modulation of different PTPs. We emphasize the Helicobacter pylori (H. pylori) virulence which is in part associated with the activation of PTP receptors. We also explore the involvement of intracellular redox state in response to H. pylori infection. In addition, some PTP members are under influence by genetic mutations, epigenetics mechanisms, and miRNA modulation. The understanding of multiple aspects of PTPs in GC may provide new targets and perspectives on drug development.

Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution.

Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, we report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a ß-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.

Epidemiology changes in peptic ulcer diseases 18 years apart explored from the genetic aspects of Helicobacter pylori.

The prevalence of peptic ulcer diseases has decreased over the past decades. The contribution of Helicobacter pylori to these changes has not been clearly delineated. Two cohorts of patients receiving esophagogastroduodenoscopy examination together with urease test were enrolled, 1 from year 2001 (n = 1030), the other from year 2019 (n = 600). The prevalence changes of peptic ulcer diseases as well as the associated clinical factors were analyzed. An independent cohort of gastric biopsy samples (n = 151) positive for H. pylori were retrieved for ureC gene genotype analysis. Comparison between the patients recruited from 2001 and 2019 revealed significant decrease in H. pylori infection (P < 0.001), duodenal ulcer prevalence (P < 0.001) and gastric ulcer prevalence (P < 0.001). Multivariate analysis showed that the decreases of these factors were independent (adjusted P < 0.001 for all). Intriguingly, in H. pylori positive patients, the prevalence of duodenal ulcer still decreased with year (P < 0.001), which was not found in gastric ulcer (P = 0.345). Genetic analysis of H. pylori urease gene showed that MboI-restriction fragment length polymorphism-defined genotype 3 UreC was significantly more prevalent in gastric ulcer patients than in others (P = 0.022). Independent decreases of H. pylori infection, gastric ulcer and duodenal ulcer over decades were found. In H. pylori positive patients, duodenal ulcer prevalence decreased overtime while gastric ulcer prevalence remained unchanged. Gastric ulcer/cancer had a higher prevalence of MboI-defined genotype 3 UreC gene.

The Gamma-glutamyltransferase gene of Helicobacter pylori can promote gastric carcinogenesis by activating Wnt signal pathway through up-regulating TET1.

AIMS: Helicobacter pylori (Hp) infection plays an important role in the development of gastric cancer. Hp can secrete gamma-glutamyltransferase (GGT), however, the impact of GGT of Hp on the human gastric cells is not clear. Although it has been demonstrated that ten-eleven translocation 1 (TET1) is overexpressed in gastric cancer, the relationship between the GGT of Hp and TET1 has not been studied. The aim of this study was to explore the relationship between GGT and TET1, and the role of TET1 in the development of gastric cancer induced by Hp was also explored. MATERIALS AND METHODS: The correlation between TET1 and prognosis of gastric adenoma cancer was analyzed by bioinformatics. The GGT gene of Hp26695 was knocked out by electroporation with plasmid to construct the GGT knockout strain of Hp (Hp-KS-1). The shTET1 lentivirus transfected GES-1, MGC-803 and SGC-7901 cell lines were constructed. The biological characteristics of the three kind of cells were detected. KEY FINDINGS: TET1 was overexpressed in gastric tissues of Hp infected patients and mice. Bioinformatics analysis showed that in patients with gastric cancer, higher TET1 expression would result in poorer prognosis. The GGT gene of Hp can lead to overexpression of TET1 in GES-1, MGC-803 and SGC-7901 cells, along with the activation of Wnt/ß-catenin signaling pathway, and then promoting tumorigenesis. After silencing TET1, the Wnt/ß-catenin signaling pathway which was activated by GGT of Hp was inhibited. SIGNIFICANCE: GGT of Helicobacter pylori can promote gastric carcinogenesis by activating Wnt/ß-catenin signaling pathway trough up-regulating TET1.

A multifunctional DNA polymerase I involves in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in Helicobacter pylori.

Helicobacter pylori is the most infectious human pathogen that causes gastritis, peptic ulcers and stomach cancer. H. pylori DNA polymerase I (HpPol I) is found to be essential for the viability of H. pylori, but its intrinsic property and attribution to the H. pylori DNA replication remain unclear. HpPol I contains a 5'→3' exonuclease (5'-Exo) and DNA polymerase (Pol) domain, respectively, but lacks a 3'→5' exonuclease, or error proofreading activity. In this study, we characterized the 5'-Exo and Pol functions of HpPol I and found that HpPol I is a multifunctional protein displaying DNA nick translation, strand-displacement synthesis, RNase H-like, structure-specific endonuclease and exonuclease activities. In the in vitro DNA replication assay, we further demonstrated that the 5'-Exo and Pol domains of HpPol I can cooperate to fill in the DNA gap, remove the unwanted RNA primer from a RNA/DNA hybrid and create a ligatable nick for the DNA ligase A of H. pylori to restore the normal duplex DNA. Altogether, our study suggests that the two catalytic domains of HpPol I may synergistically play an important role in the maturation of Okazaki fragments during the lagging-strand DNA synthesis in H. pylori. Like the functions of DNA polymerase I in Escherichia coli, HpPol I may involve in both DNA replication and repair in H. pylori.

Synthesis and Structure-Activity Relationship Studies of N-monosubstituted Aroylthioureas as Urease Inhibitors.

BACKGROUND: Thiourea is a classical urease inhibitor which is usually used as a positive control, and many N,N'-disubstituted thioureas have been determined as urease inhibitors. However, due to steric hindrance, N,N'-disubstituted thiourea motif could not bind urease as thiourea. On the contrary, N-monosubstituted thiourea with a tiny thiourea motif could theoretically bind into the active pocket as thiourea. OBJECTIVE: A series of N-monosubstituted aroylthioureas were designed and synthesized for evaluation as urease inhibitors. METHODS: Urease inhibition was determined by the indophenol method and IC50 values were calculated using computerized linear regression analysis of quantal log dose-probit functions. The kinetic parameters were estimated via surface plasmon resonance (SPR) and by nonlinear regression analysis based on the mixed type inhibition model derived from Michaelis-Menten kinetics. RESULTS: Compounds b2, b11, and b19 reversibly inhibited urease with a mixed mechanism, and showed excellent potency against both cell-free urease and urease in the intact cell, with IC50 values being 90- to 450-fold and 5- to 50-fold lower than the positive control acetohydroxamic acid, respectively. The most potent compound b11 showed an IC50 value of 0.060 ± 0.004µM against cell-free urease, which bound to urea binding site with a very low KD value (0.420±0.003nM) and a very long residence time (6.7 min). Compound b11 was also demonstrated to have very low cytotoxicity to mammalian cells. CONCLUSION: The results revealed that N-monosubstituted aroylthioureas bound to the active site of urease as expected, and represent a new class of urease inhibitors for the development of potential therapeutics against infections caused by urease-containing pathogens.