Association of LEC and tnpA Helicobacter pylori genes with gastric cancer in a Brazilian population

Abstract Background H. pylori seroprevalence in Brazilians varies and is dependent on socioeconomic status, sanitation conditions and ethnicity; furthermore, H. pylori is not always associated with the incidence of gastric cancer, suggesting the role of more virulent strains. The purpose of this study was to analyze the association of more virulent H. pylori strains with gastric cancer. Methods DNA was extracted from gastric biopsies of thirty-four cases of gastric cancer (11 intestinal-type, 23 diffuse-type), and thirty-four of patients with endoscopic gastritis. The presence of cagPAI genes (cagA, cagA promoter, cagE, cagM, tnpB, tnpA, cagT and the left end of the cagII (LEC)) and babA were analyzed by PCR. Results Comparison of H. pylori isolates from gastric cancer and gastritis patients showed significant associations of tnpA and LEC with gastric cancer (73.5% [OR, 6.66; 95% CI, 2.30-19.25] and 58.8% [OR, 10.71; 95% CI, 3.07-37.28] of cases, respectively). Other cagPAI genes were detected in both groups at similar frequencies. Conclusions tnpA and LEC of H. pylori cagPAI were associated with gastric cancer; nonetheless, these results were restricted within this group of patients and further studies are needed to confirm these results in a larger sample and determine their role in gastric carcinogenesis.

Analisi del circuito di regolazione responsabile del controllo trascrizionale dei geni Heat-Shock in Helicobacter pylori

Helicobacter pylori, un patogeno umano in grado di colonizzare la nicchia gastrica, è associato a patologie del tratto gastrointestinale di varia gravità. Per sopravvivere nell’ambiente ostile dello stomaco dell’ospite, e mettere in atto un’infezione persistente, il batterio si serve di una serie di fattori di virulenza che includono anche le proteine Heat Shock (chaperone). I principali geni codificanti le proteine chaperone in H. pylori sono organizzati in tre operoni trascritti dall’RNA polimerasi contenente il fattore sigma vegetativo σ80. La trascrizione di due dei tre operoni è regolata negativamente da due regolatori trascrizionali, HspR e HrcA, mentre il terzo operone è represso solo da HspR. Fino ad ora, studi molecolari per la comprensione del ruolo di ciascuna proteina nel controllo trascrizionale dei geni heat shock sono stati ostacolati dalla citotossicità ed insolubilità di HrcA quando espressa in sistemi eterologhi. In questo lavoro, è stata analizzata la sequenza amminoacidica di HrcA ed è stata confermata sperimentalmente la predizione bioinformatica della sua associazione con la membrana interna. La citotossicità e l’insolubilità di HrcA in E. coli sono state alleviate inducendone l’espressione a 42°C. Saggi in vitro con le proteine ricombinanti purificate, HspR e HrcA, hanno consentito di definire i siti di legame dei due repressori sui promotori degli operoni heat shock. Ulteriori saggi in vitro hanno suggerito che l’affinità di HrcA per gli operatori è aumentata dalla chaperonina GroESL. Questi dati contribuiscono parzialmente alla comprensione del meccanismo di repressione della trascrizione espletato da HrcA e HspR e permettono di ipotizzare il coinvolgimento di altri regolatori trascrizionali. L’analisi di RNA estratti dal ceppo selvatico e dai mutanti hrcA, hspR e hrcA/hspR di H.pylori su DNAmacroarrays non ha evidenziato il coinvolgimento di altri regolatori trascrizionali, ma ha permesso l’identificazione di un gruppo di geni indotti da HrcA e/ HspR. Questi geni sono coinvolti nella biosintesi e regolazione dell’apparato flagellare, suggerendo un’interconnessione tra la risposta heat shock e la motilità e chemiotassi del batterio.

Molecular architecture of fur binding to iron-induced and - repressed genes in Helicobacter pylori

The ferric uptake regulator protein Fur regulates iron-dependent gene expression in bacteria. In the human pathogen Helicobacter pylori, Fur has been shown to regulate iron-induced and iron-repressed genes. Herein we investigate the molecular mechanisms that control this differential iron-responsive Fur regulation. Hydroxyl radical footprinting showed that Fur has different binding architectures, which characterize distinct operator typologies. On operators recognized with higher affinity by holo-Fur, the protein binds to a continuous AT-rich stretch of about 20 bp, displaying an extended protection pattern. This is indicative of protein wrapping around the DNA helix. DNA binding interference assays with the minor groove binding drug distamycin A, point out that the recognition of the holo-operators occurs through the minor groove of the DNA. By contrast, on the apo-operators, Fur binds primarily to thymine dimers within a newly identified TCATTn10TT consensus element, indicative of Fur binding to one side of the DNA, in the major groove of the double helix. Reconstitution of the TCATTn10TT motif within a holo-operator results in a feature binding swap from an holo-Fur- to an apo-Fur-recognized operator, affecting both affinity and binding architecture of Fur, and conferring apo-Fur repression features in vivo. Size exclusion chromatography indicated that Fur is a dimer in solution. However, in the presence of divalent metal ions the protein is able to multimerize. Accordingly, apo-Fur binds DNA as a dimer in gel shift assays, while in presence of iron, higher order complexes are formed. Stoichiometric Ferguson analysis indicates that these complexes correspond to one or two Fur tetramers, each bound to an operator element. Together these data suggest that the apo- and holo-Fur repression mechanisms apparently rely on two distinctive modes of operator-recognition, involving respectively the readout of a specific nucleotide consensus motif in the major groove for apo-operators, and the recognition of AT-rich stretches in the minor groove for holo-operators, whereas the iron-responsive binding affinity is controlled through metal-dependent shaping of the protein structure in order to match preferentially the major or the minor groove.

Transcriptional responses of the Helicobacter pylori cag pathogenicity island

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

Secondary analysis of effectivness of tinidazole-containing-quadruple eradication therapy in asymptomatic H. pylori infected children in El Paso

Helicobacter pylori infection is frequently acquired during childhood. This microorganism is known to cause gastritis, and duodenal ulcer in pediatric patients, however most children remain completely asymptomatic to the infection. Currently there is no consensus in favor of treatment of H. pylori infection in asymptomatic children. The firstline of treatment for this population is triple medication therapy including two antibacterial agents and one proton pump inhibitor for a 2 week duration course. Decreased eradication rate of less than 75% has been documented with the use of this first-line therapy but novel tinidazole-containing quadruple sequential therapies seem worth investigating. None of the previous studies on such therapy has been done in the United States of America. As part of an iron deficiency anemia study in asymptomatic H. pylori infected children of El Paso, Texas, we conducted a secondary data analysis of study data collected in this trial to assess the effectiveness of this tinidazole-containing sequential quadruple therapy compared to placebo on clearing the infection. Subjects were selected from a group of asymptomatic children identified through household visits to 11,365 randomly selected dwelling units. After obtaining parental consent and child assent a total of 1,821 children 3-10 years of age were screened and 235 were positive to a novel urine immunoglobulin class G antibodies test for H. pylori infection and confirmed as infected using a 13C urea breath test, using a hydrolysis urea rate >10 μg/min as cut-off value. Out of those, 119 study subjects had a complete physical exam and baseline blood work and were randomly allocated to four groups, two of which received active H. pylori eradication medication alone or in combination with iron, while the other two received iron only or placebo only. Follow up visits to their houses were done to assess compliance and occurrence of adverse events and at 45+ days post-treatment, a second urea breath test was performed to assess their infection status. The effectiveness was primarily assessed on intent to treat basis (i.e., according to their treatment allocation), and the proportion of those who cleared their infection using a cut-off value >10 μg/min of for urea hydrolysis rate, was the primary outcome. Also we conducted analysis on a per-protocol basis and according to the cytotoxin associated gene A product of the H. pylori infection status. Also we compared the rate of adverse events across the two arms. On intent-to-treat and per-protocol analyses, 44.3% and 52.9%, respectively, of the children receiving the novel quadruple sequential eradication cleared their infection compared to 12.2% and 15.4% in the arms receiving iron or placebo only, respectively. Such differences were statistically significant (p<0.001). The study medications were well accepted and safe. In conclusion, we found in this study population, of mostly asymptomatically H. pylori infected children, living in the US along the border with Mexico, that the quadruple sequential eradication therapy cleared the infection in only half of the children receiving this treatment. Research is needed to assess the antimicrobial susceptibility of the strains of H. pylori infecting this population to formulate more effective therapies. ^

Free recombination within Helicobacter pylori

Sequences of three gene fragments (flaA, flaB, and vacA) from Helicobacter pylori strains isolated from patients in Germany, Canada, and South Africa were analyzed for diversity and for linkage equilibrium by using the Homoplasy Test and compatibility matrices. Horizontal genetic exchange in H. pylori is so frequent that different loci and polymorphisms within each locus are all at linkage equilibrium. These results indicate that H. pylori is panmictic. Comparisons with sequences from Escherichia coli, Neisseria meningitidis, and Drosophila melanogaster showed that recombination in H. pylori was much more frequent than in other species. In contrast, when multiple family members infected with H. pylori were investigated, some strains were indistinguishable at all three loci. Thus, H. pylori is clonal over short time periods after natural transmission.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Altered states: Involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori

Helicobacter pylori, present in half of the world’s population, is a very successful pathogen. It can survive for decades in the human stomach with few obvious consequences to the host. However, it is also the cause of gastric diseases ranging from gastritis to ulcers to gastric cancer and has been classified a type 1 carcinogen by the World Health Organization. We have previously shown that phosphorylation of a 145-kDa protein and activation of signal transduction pathways are associated with the attachment of H. pylori to gastric cells. Here we identify the 145-kDa protein as the H. pylori CagA protein. We also show that CagA is necessary to induce a growth-factor-like phenotype (hummingbird) in host gastric cells similar to that induced by hepatocyte growth factor (HGF). Additionally, we identify a second cellular phenotype induced after attachment by H. pylori, which we call SFA (stress fiber associated). SFA is CagA independent and is produced by type I and type II H. pylori.

cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3′ end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH ≈5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.