Pathogenic Bacterium Helicobacter Pylori Research Articles

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.

The pursuit of new alternative ways to eradicate Helicobacter pylori continues: Detailed characterization of interactions in the adenylosuccinate synthetase active site

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287–293 and 40–44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40–50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori.

Ligustrazine Attenuates Gastric Inflammation and Apoptosis in Helicobacter pylori-induced Gastric Epithelial Cells

Background: Stomach disorders, including gastric cancer and gastritis, are associated with the pathogenic bacterium Helicobacter pylori. Enhanced inflammation is the characteristic of H. pylori-induced gastritis. Ligustrazine exerts anti-inflammatory properties in mouse asthma models and acute kidney injury. Objectives: To determine the role of ligustrazine in H. pylori-induced gastritis. Methods: Normal gastric epithelial cell line (GES-1) was cultured with H. pylori at a multiplicity of infection (MOI) of 100: 1 for 24 hours. GES-1 cell line under H. pylori condition was incubated with 100 or 200 μM ligustrazine for 24 hours. Cell viability and apoptosis were investigated by MTT and flow cytometry assays, respectively. Inflammation was assessed by determining the levels and mRNA expression of interleukins (IL)-6/8, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein 1 (MCP-1) using ELISA and qRT-PCR analysis, respectively. Results: Helicobacter pylori infection reduced the viability and promoted the apoptosis of GES-1 cell line, accompanied by the enhanced activities of caspases 3 and 9. However, ligustrazine reversed the H. pylori-induced infection decreased viability, while increased apoptosis and caspases 3/9 activities in GES-1 cell line. Moreover, ligustrazine attenuated H. pylori-induced secretions of pro-inflammatory factors, IL-6/8, TNF-α, and MCP-1, in GES-1 cell line. The protein expression of inhibitor of NF-κB (IκBα) was downregulated in GES-1 cell line after H. pylori infection, while the protein expression levels of p65 and phosphorylation of IκBα were upregulated by H. pylori infection. On the contrary, ligustrazine decreased H. pylori-induced protein expression of IκBα, whereas increased protein expression of p65 and phosphorylation of IκBα. Conclusions: Ligustrazine exerted protective effects on H. pylori-induced gastric epithelial cells through inhibition of gastric inflammation and apoptosis and inactivation of NF-κB pathway.

Improving Phenolic-Linked Antioxidant, Antihyperglycemic and Antibacterial Properties of Emmer and Conventional Wheat Using Beneficial Lactic Acid Bacteria

Beneficial lactic acid bacteria (LAB)-based fermentation is an effective bioprocessing approach to improve human-health-targeted functional benefits of plant-based food substrates, such as cereal grains. Previously, we observed high phenolic bioactive-linked antioxidant and anti-hyperglycemic properties in whole grain Emmer (hulled). In this study, beneficial LAB (Lactiplantibacillus plantarum) was recruited to ferment (0–72 h) aqueous extracts (0.4 g/mL concentration) of previously optimized hulled Emmer wheat and conventional red spring wheat cv. Barlow. The fermented and unfermented (control) wheat extracts were analyzed for phenolic content, phenolic profile, antioxidant activity, and antihyperglycemic properties (α-amylase and α-glucosidase enzyme inhibitory activity) using in vitro assay models. Additionally, antimicrobial activity against pathogenic bacteria Helicobacter pylori, and potential prebiotic activity supporting the growth of beneficial Bifidobacterium longum were also investigated. Improvement in antioxidant activity and antihyperglycemic functional benefits were observed, while soluble phenolic content remained high after 72 h fermentation. Antimicrobial activity against H. pylori was also observed in 48 and 72 h fermented wheat extracts. This study provides an insight into the efficacy of LAB-based fermentation as a safe bioprocessing tool to design health-targeted functional foods and ingredients from underutilized whole grains like Emmer for targeting type 2 diabetes dietary benefits.

Four Chromosomal Type IV Secretion Systems in Helicobacter pylori: Composition, Structure and Function.

The pathogenic bacterium Helicobacter pylori is genetically highly diverse and a major risk factor for the development of peptic ulcer disease and gastric adenocarcinoma in humans. During evolution, H. pylori has acquired multiple type IV secretion systems (T4SSs), and then adapted for various purposes. These T4SSs represent remarkable molecular transporter machines, often associated with an extracellular pilus structure present in many bacteria, which are commonly composed of multiple structural proteins spanning the inner and outer membranes. By definition, these T4SSs exhibit central functions mediated through the contact-dependent conjugative transfer of mobile DNA elements, the contact-independent release and uptake of DNA into and from the extracellular environment as well as the secretion of effector proteins in mammalian host target cells. In recent years, numerous features on the molecular functionality of these T4SSs were disclosed. H. pylori encodes up to four T4SSs on its chromosome, namely the Cag T4SS present in the cag pathogenicity island (cagPAI), the ComB system, as well as the Tfs3 and Tfs4 T4SSs, some of which exhibit unique T4SS functions. The Cag T4SS facilitates the delivery of the CagA effector protein and pro-inflammatory signal transduction through translocated ADP-heptose and chromosomal DNA, while various structural pilus proteins can target host cell receptors such as integrins or TLR5. The ComB apparatus mediates the import of free DNA from the extracellular milieu, whereas Tfs3 may accomplish the secretion or translocation of effector protein CtkA. Both Tfs3 and Tfs4 are furthermore presumed to act as conjugative DNA transfer machineries due to the presence of tyrosine recombinases with cognate recognition sequences, conjugational relaxases, and potential origins of transfer (oriT) found within the tfs3 and tfs4 genome islands. In addition, some extrachromosomal plasmids, transposons and phages have been discovered in multiple H. pylori isolates. The genetic exchange mediated by DNA mobilization events of chromosomal genes and plasmids combined with recombination events could account for much of the genetic diversity found in H. pylori. In this review, we highlight our current knowledge on the four T4SSs and the involved mechanisms with consequences for H. pylori adaptation to the hostile environment in the human stomach.

Peptides Containing meso-Oxa-Diaminopimelic Acid as Substrates for the Cell-Shape-Determining Proteases Csd6 and Pgp2.

The enzymes Csd6 and Pgp2 are peptidoglycan (PG) proteases found in the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, respectively. These enzymes are involved in the trimming of non-crosslinked PG sidechains and catalyze the cleavage of the bond between meso-diaminopimelic acid (meso-Dap) and d-alanine, thus converting a PG tetrapeptide into a PG tripeptide. They are known to be cell-shape-determining enzymes, because deletion of the corresponding genes results in mutant strains that have lost the normal helical phenotype and instead possess a straight-rod morphology. In this work, we report two approaches directed towards the synthesis of the tripeptide substrate Ac-iso-d-Glu-meso-oxa-Dap-d-Ala, which serves as a mimic of the terminus of an non-crosslinked PG tetrapeptide substrate. The isosteric analogue meso-oxa-Dap was utilized in place of meso-Dap to simplify the synthetic procedure. The more efficient synthesis involved ring opening of a peptide-embedded aziridine by a serine-based nucleophile. A branched tetrapeptide was also prepared as a mimic of the terminus of a crosslinked PG tetrapeptide. We used MS analysis to demonstrate that the tripeptide serves as a substrate for both Csd6 and Pgp2 and that the branched tetrapeptide serves as a substrate for Pgp2, albeit at a significantly slower rate.

Analysis of Helicobacter pylori Prevalence in Chittagong, Bangladesh, Based on PCR and CLO Test.

The pathogenic bacterium Helicobacter pylori is a causative agent of gastric diseases in Bangladesh as well as throughout the world. This study aimed at analyzing the prevalence of H. pylori infection among dyspeptic patients in Chittagong, the second most populous city of Bangladesh, using 16S rRNA-based H. pylori-specific Polymerase Chain Reaction and Campylobacter-like organism test. We found that 67% of the population under study was positive for H. pylori infection. Gastric ulcer and duodenal ulcer disease showed statistically significant association with H. pylori infection; however, no association of H. pylori infection was observed in terms of age and gender. This study would play a crucial role in managing H. pylori-induced gastric diseases by understanding the current trend of H. pylori infection in the Chittagong region of Bangladesh.

Discovery of an asymmetric ATPase with two catalytic centers that are separately activated by two different misacylated‐tRNAs (751.2)

A majority of microorganisms lack a complete set of aminoacyl‐tRNA synthetases but still utilize the twenty standard amino acids in their proteomes. The pathogenic bacterium Helicobacter pylori is missing both the glutaminyl‐ and asparaginyl‐tRNA synthetases and instead uses indirect tRNA aminoacylation to generate Gln‐tRNAGln and Asn‐tRNAAsn. This process proceeds via misacylation of these tRNAs to produce Glu‐tRNAGln and Asp‐tRNAAsn, followed by transamidative conversion of the two amino acid side chains to produce Gln‐tRNAGln and Asn‐tRNAAsn. Both misacylated tRNA conversions rely on an amidotransferase called GatCAB. We have identified a new enzyme from H. pylori that induces the formation of a macromolecular complex that also contains this organism’s misacylating aspartyl‐tRNA synthetase (ND‐AspRS) and GatCAB. This enzyme contains two different ATPase active sites, one based on a classic P‐loop ATPase domain and the second based on an [[Unsupported Character ‐ Symbol Font □]]‐adenosine nucleotide hydrolase (AANH) domain. Remarkably, the P‐Loop ATPase is activated by binding of Asp‐tRNAAsn, whereas the AANH ATPase is activated by Glu‐tRNAGln. These ATPase activities modulate GatCAB activity to drive conversion of both misacylated tRNAs into their cognate products. The asymmetric nature of this enzyme and its activation by different misacylated tRNAs make it an unusual new participant in tRNA aminoacylation and protein translation.

Intracellular Locations of Replication Proteins and the Origin of Replication during Chromosome Duplication in the Slowly Growing Human Pathogen Helicobacter pylori

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

Latin America: ripe for cutting-edge research proposals for prevention and control of Helicobacter pylori

One of the greatest areas of scientific progress has been the identification of certain infectious agents that are implicated in the carcinogenic process. This new knowledge has had broad repercussions not only in the application of new primary prevention options, such as prophylactic vaccines, but also in the definition of infection biomarkers that can be used as screening alternatives or as prognostic markers for neoplasia. We now know that chronic infection with the pathogenic bacteria Helicobacter pylori (H. pylori) is associated with gastrointestinal tract disorders, ranging from chronic gastritis to gastric adenocarcinoma; there is also evidence of a possible association with gastric lymphoma and peptic ulcer. Published reports support the existence of geographic regions where this bacterium is endemic and where H. pylori colonization occurs at a very early age in populations living in precarious sanitary conditions. For this reason, gastric cancer is one of the most common cancers in the world in low-resource countries and, as noted in the accompanying articles [1, 2], one with great heterogeneity in terms of incidence and mortality in Latin America. As a result, this geographic region constitutes a fascinating setting for research and provides unique and important opportunities for gastric cancer prevention. From a public health perspective, there are currently two major lines of research and action in regard to H. pylori and gastric cancer. On the one hand, we should gain a more precise understanding of the natural history of H. pylori infection and, as a consequence, learn about the triggering mechanism(s) related to chronic exposure in the development of gastric adenocarcinoma. However, we also know that reducing future incidence of gastric cancer is possible by improving the living conditions of the general population from a hygienic perspective, as confirmed by Porras et al. [3] in this special edition. Evidence now exists, which established H. pylori as a part of normal human microbiota [4]. This line of work indicates that this bacterium has a special tropism for gastric mucosa, that strains with toxic potential exist, and that the bacteria secrete proteins such as vacA [5], which produce vacuolization in cultures of human epithelial cells as well as increased capacity to modify molecules. Given the existing evidence from biomedical and epidemiological research underscoring the high degree of genetic diversity in H. pylori [6], prevention is arguably very complex. Research groups in Latin America have generated an invaluable scientific tradition for generation of new hypotheses [7], evidence for interventions [8], and high-impact publications [9] related to the study of the carcinogenic process in gastroduodenal mucosa. Recently, a Latin American H. pylori research consortium was formed with the primary mission of evaluating effectiveness, feasibility, and cost of eradication using an antimicrobial regimen specifically for Latin America. Results are encouraging and demonstrate the technical and academic abilities of the participating groups [10]. They also indicate that treatment regimens for successful eradication need to be region-specific; thus, adopting successful treatment regimens from high-income countries may not be the best solution for lower-income countries. Adding to this line of work, results of a diverse set of research studies conducted in various regions of Latin America are presented in this special issue, which reveal the great heterogeneity that exists in the region. These publications include topics such as potential solutions for gastric cancer surveillance in low-resource countries [11], ecological data in support of the altitude–gastric cancer association [1], assessment of the utility of serologic biomarkers for risk of gastric cancer in three Latin American countries [2], confirmation of risk factors for H. pylori infection [3], and a meta-analysis of a variety of risk factors and gastric cancer risk [12]. The work presented in this special edition of the journal along with the recently formed consortium underscores the enormous potential that multi-centric regional studies have for addressing the magnitude of health problems, and even more important, identify solutions for prevention of future disease. These invaluable resources allow us to postulate new research hypotheses and to consolidate a regional group, which can contribute to improving therapeutic strategies for prevention and control of gastric cancer and build the foundation for evidence-based regional public policies aimed at improve the quality of life of the population.

Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children

BackgroundThe protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.Methodology/Principal FindingsFecal samples from 427 apparently healthy children, 0–12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7–4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection.Conclusions/SignificanceThis study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.

Pathogenic bacterium Helicobacter pylori alters the expression profile of p53 protein isoforms and p53 response to cellular stresses

The p53 protein plays a central role in the prevention of tumorigenesis. Cellular stresses, such as DNA damage and aberrant oncogene activation, trigger induction of p53 that halts cellular proliferation and allows cells to be repaired. If cellular damage is beyond the capability of the repair mechanisms, p53 induces apoptosis or cell cycle arrest, preventing damaged cells from becoming cancerous. However, emerging evidence suggests that the function of p53 needs to be considered as isoform-specific. Here, we report that the expression profile of p53 can be shifted toward inhibitory p53 isoforms by the pathogenic bacterium Helicobacter pylori, which is known for its strong association with gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. We found that interaction of H. pylori with gastric epithelial cells, mediated via the cag pathogenicity island, induces N-terminally truncated Δ133p53 and Δ160p53 isoforms in human cells. Induction of an orthologous p53 isoform, Δ153p53, was also found in H. pylori-infected Mongolian gerbils. The p53 isoforms inhibit p53 and p73 activities, induce NF-κB, and increase survival of infected cells. Expression of Δ133p53, in response to H. pylori infection, is regulated by phosphorylation of c-Jun and activation of activator protein-1-dependent transcription. Together, these results provide unique insights into the regulation of p53 protein and may contribute to the understanding of tumorigenesis associated with H. pylori.

Novel probiotic Bifidobacterium bifidum CECT 7366 strain active against the pathogenic bacterium Helicobacter pylori.

Helicobacter pylori is considered one of the major risk factors underlying the development of gastritis and gastric and duodenal ulcers. Moreover, 50% of the population carries this bacterium, and consequently, when it is detected, eradication of H. pylori is strongly recommended. Regarding the use of probiotics as functional agents, several studies have shown that there is a direct relationship between the addition of certain probiotic bacteria and in vitro inhibition of H. pylori; however, in vivo studies showing bifidobacterial activity against H. pylori remain scarce. In this study, a Bifidobacterium bifidum strain which proved active in vitro against H. pylori has been isolated, with inhibition levels reaching 81.94% in the case of the supernatant and even 94.77% inhibition for supernatant purified by cationic exchange followed by an inverse phase. In vivo studies using a BALB/c mouse model have proved that this strain partially relieves damage to gastric tissues caused by the pathogen and also decreases the H. pylori pathogenicity ratio. This novel strain fulfills the main properties required of a probiotic (resistance to gastrointestinal juices, biliary salts, NaCl, and low pH; adhesion to intestinal mucus; and sensitivity to antibiotics). Furthermore, the absence of undesirable metabolites has been demonstrated, and its food safety status has been confirmed by acute ingestion studies in mice. In summary, the results presented here demonstrate that Bifidobacterium bifidum CECT 7366 can be considered a probiotic able to inhibit H. pylori both in vitro and in vivo.

Cloning of a Cholesterol-α-glucosyltransferase from Helicobacter pylori

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

Crystallization and preliminary X-ray crystallographic analysis of UDP-N-acetylglucosamine acyltransferase from Helicobacter pylori.

Lipid A, a constituent of lipopolysaccharides, is essential for the growth and virulence of most Gram-negative bacteria. This makes its biosynthetic enzymes potential targets for development of new antibacterial agents. The first step of lipid A biosynthesis is catalyzed by the enzyme UDP-N-acetylglucosamine acyltransferase (LpxA). LpxA from the pathogenic bacterium Helicobacter pylori has been overexpressed in Escherichia coli and crystallized at 297 K using ammonium sulfate and sodium/potassium tartrate as precipitants in the presence of a detergent. Diffraction data to 2.1 A resolution have been collected from a native crystal. The crystal belongs to space group P6(3)22, with unit-cell parameters a = b = 90.69, c = 148.20 A. The asymmetric unit contains one subunit of LpxA, with a crystal volume per protein mass (V(M)) of 2.87 A(3) Da(-1) and a solvent content of 57.1%.

Crystallization and preliminary X-ray crystallographic analysis of aspartate 1-decarboxylase from Helicobacter pylori.

Aspartate 1-decarboxylase (PanD) catalyzes the alpha-decarboxylation of L-aspartate in the major route of beta-alanine production for pantothenate biosynthesis in bacteria. Pantothenate is synthesized in microorganisms, plants and fungi but not in animals and thus the enzymes of its biosynthetic pathway are potential targets for developing agents against these organisms. PanD from the pathogenic bacterium Helicobacter pylori has been overexpressed in Escherichia coli and crystallized using sodium formate as a precipitant. Crystals diffracted to better than 1.5 A Bragg spacing upon exposure to synchrotron X-rays. Diffraction data to 1.55 A have been collected from a crystal grown in the presence of the substrate analogue isoasparagine. The crystal belongs to the tetragonal space group I422, with unit-cell parameters a = b = 81.83, c = 93.78 A. The asymmetric unit contains one subunit of PanD, with a corresponding crystal volume per protein mass (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 56.8%.

Disinfectant activity against different morphological forms of Helicobacter pylori: first results

The pathogenic bacterium Helicobacter pylori, which has infected more than one-half of the world's human population, exists in two morphological forms; the viable helical form and the disputed viable-but-not-culturable coccoid form. Infection by the helical form proceeds through the oral-oral route, while that by the coccoid form, if possible at all, is by the faecal-oral and/or the oral-oral route. The present pilot study addresses the question of disinfectant efficacy against both forms of the bacterium.

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).

Helicobacter pylori interacts with heparin and heparin-dependent growth factors.

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 x 10(-12)M), and also heparan sulfate and heparin with higher affinity (Kd 9 x 10(-9)M) than FGFs bind to heparin (10(-8) - 10(-9)M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanisms. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.

Helicobacter pylori interacts with heparin and heparin-dependent growth factors

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 × 10 −12 M), and also heparan sulfate and heparin with higher affinity ( K d 9 × 10 −9 M) than FGFs bind to heparin (10 −8–10 −9 M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanism. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.