41 research outputs found

    Assessment of anti-urease and consequential inhibitory potential of South African honey extracts on the multiplication of drug-resistant, vacA and cagA positive helicobacter pylori strains under acidic conditions

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    Helicobacter pylori, a neutralophile chronically infects the gastric stomach of more than half of the world’s population. Infection with the organism is associated with acute or chronic duodenal/gastric ulcer disease, gastritis, gastric adenocarcinoma, mucosa-associated tissue lymphoma (MALT) and primary B-cell gastric lymphoma and has been grouped as a class one carcinogen by the World Health Organisation (WHO). Prevalence of this organism is very high in developing countries especially in Africa, including South Africa. H. pylori treatment using the common first and second line regimens, triple therapy with two antibiotics and a proton pump inhibitor (PPI) is showing inefficiency due to increasing drug resistance. However, newly developed treatment regimens seem to be more expensive and are accompanied by more side effects. Honey contains phytochemicals which are a wealthy source of biologically active compounds some of which have been put into good use in the pharmaceutical industry. Pathogenesis of H. pylori infection in the human stomach relies on several virulence factors which include the urease enzyme, cagA and vacA. The urease enzyme actively hydrolyses urea to produce ammonia an important by-product involved in pH regulation favouring the survival of the organism in the acidic human stomach. This study therefore focuses on screening for anti-urease solvent extracts of South African honey, and evaluate whether inhibition of urease offsets the growth of H. pylori under acidic conditions. Locally produced natural honeys; Bush honey, Raw honey, Gold Crest honey, Q Bee honey, Little Bee honey, Fleures honey-radurised, Siyakholwa pure honey and Manuka honey; an import from New Zealand were purchased and the method by Syazana et al. (2010) was used for the extraction of compounds in honey. A standard strain ATCC 43526 (American Type Culture Collection, Manassas, VA, USA) and 48 pure cultures obtained from clinical isolates cultured from gastric corpus biopsy specimen of patients with gastric morbidities who were ix visiting the endoscopy unit in Livingstone Hospital, Port Elizabeth between June 2008 to December 2008 were initially used as source of urease enzyme as per extraction method done by Amin et al. (2013), but with modifications. Prior to urease extraction, H. pylori strains were identified by biochemical tests (urease, catalase, oxidase, Gram stain), confirmed by PCR targeting the glmM gene (140 bp) and drug resistance profiling was done on all the 48 strains according to Seanego et al. (2012). The screening for anti-urease active compounds was done according to Kaltwasser et al. (1966), a method relying on the reduction of NADH in a coupled urease dehydrogenase (GDH) system. Acetohydroxamic acid was used as a standard inhibitor. Prevalence of cytotoxin-associated gene A (cagA) gene and vacuolating cytotoxin gene A (vacA) gene was determined among all 48 clinical samples. The standard strains of H. pylori, X47 (cagA positive), J99 (vacA s1m1) and Tx30a (s2m2) were used as positive controls. H. pylori’s growth was then monitored under acidic pH in a cocktail spiked with anti-urease compounds (test samples) and in a cocktail without anti-urease compounds (negative control). Acetohydroxamic acid was used as a standard urease inhibitor. H. pylori multiplication was monitored in Brain Heart Infusion Broth (BHIB) adjusted to pH of 2, 3, 4, 5, 6 and 7. The strain MP01 was used as a standard urease negative strain while X47 and J99 were used as positive standards for cagA and vacA s1m1 respectively. The compounds that had anti-urease activity and were successful towards suppressing the multiplication of H. pylori under acidic environment, all other factors optimised, were subjected to gas chromatograph mass spectrometry (GC-MS) and liquid chromatograph spectrometry (LCMS) to determine volatile compounds and drugs in honey extracts respectively. The findings of this study revealed that at a concentration of 50 mg/mL, urease inhibition by petroleum ether extracts of Gold Crest and Fleures honey, hexane extracts of Little Bee and Manuka honey, and chloroform extracts of Bush honey and Q Bee honey had a range above or equal to 50 percent and there was no significance difference in urease inhibition percentage (I percent) of urease from different sources including that extracted from drug resistant H. pylori (p >0.05). Virulence factors are important for the pathogenesis of H. pylori. All the 48 clinical isolates were glmM (140 bp) positive and cagA was detected in 97.9 percent of the test isolates. The vacA gene was detected in all isolates but with different subtypes. The vacA allelic combination s1m1 was detected in 75 percent of the test isolates and s1m2 allelic combination was detected in 16.7 percent of the test isolates while the combination s2m2 was detected in 8.3 percent of the test isolates. None of the test isolates possessed the allelic combination s2m1. When H. pylori multiplication was monitored under acidic conditions in the presence of anti-urease active compounds, it was revealed that anti-urease active compounds in honey are capable of inhibiting the normal multiplication of H. pylori strains that are cagA positive, vacA positive and drug resistant. The GC-MS analysis showed that Fleures honey (urease I percent = 67.8 – 68.5 percent) and Gold Crest honey (urease I percent = 50.9 percent – 53.3 percent), all petroleum ether extracts had 27 and 26 volatile compounds. The hexane extract of Manuka honey (urease I percent = 50.0 – 53.2) had 43 compounds detected. The chloroform extract of Q Bee (urease I percent = 64.2 – 66.2 percent) had 13 volatile compounds detected. All the volatile compounds considered as representative samples of GC-MS analysis had a spectral matching ≥ 90 percent with the NIST11 library. However, the majority of compounds that were detected by LC-MS in representative honey extracts include vardenafil, urapidil, hydrocortisone, e.t.c which are drugs commonly used in the treatment of different ailments or infections that affect human beings. In addition, two xi drugs, sulfaquinoxaline and hydroxyquinoline which are used in veterinary medicine and antiseptic, disinfectant and pesticide applications in agricultural activities were detected in Little Bee honey. We therefore conclude that inhibition of urease has a bactericidal effect on drug resistant, cagA positive and vacA positive H. pylori strains growing under acidic environment

    Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

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    Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylor

    Identification of an immunogenic 18 kDa protein of Helicobacter pylori using alkaline phosphatase gene fusions : a thesis presented in partial fulfillment of the requirement for the Doctor of Philosophy in Molecular Microbiology, Massey University, Palmerston North, NZ

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    Secreted or surface-associated proteins play an important role in the immunopathogenesis of Helicobacter pylori infection. The aim of this study was to identify, using a genetic approach, H. pylori exported proteins and assess their role in the host immune response to infection. As part of this work, an H. pylori expression library was constructed and screened with a monoclonal antibody raised to a component of outer membrane vesicles from H. pylori, identified and characterised in a separate study. The screening strategy identified a locus of the genome containing two genes encoding exported proteins. Subsequent expression studies identified the gene product detected by the antibody as Lpp20, which encodes a well characterised lipoprotein from H. pylori. In addition, the use of alkaline phosphatase (AP) gene fusion methodology enabled the identification of a large number of other H. pylori exported proteins. Immunoscreening of a selection of enzymatically active H. pylori AP fusion proteins was carried out by Western blot analysis with patient sera and lymphocyte proliferation assays using peripheral blood mononuclear cells from H. pylori infected individuals. These assays identified a novel H. pylori exported antigen which was recognised by antibody derived from H. pylori infected individuals. Southern blot analysis revealed that the gene encoding the protein was absent in other Helicobacter species tested and sequence analysis of the gene demonstrated that it is highly conserved among H. pylori isolates. In order to obtain pure recombinant protein, the gene encoding the protein was cloned and expressed as a Beta-galactosidase (β-gal) fusion in Escherichia coli and the protein purified by affinity chromatography. The size of the recombinant protein released (18 kDa) was consistent with the calculated molecular mass of the polypeptide deduced from the DNA sequence. In Western blot assays, the purified protein was recognised by 71% of sera taken from patients infected with H. pylori, but by only 16% of sera taken from patients with unrelated or with no gastrointestinal disease. These results indicated that the 18 kDa protein from H. pylori was immunogenic and expressed in vivo. In other experiments, it was found that oral administration of this antigen did not protect mice against H. pylori colonisation following challenge with H. pylori

    Activities of Muscadine Grape Skin and Polyphenolic Constituents against Helicobacter pylori

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    Helicobacter pylori is a microaerophilic, gram-negative bacterium and among the most persistent of all human pathogens. Its presence in the gastric environment is corre-lated with diseases such as gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. Multiple factors are believed responsible for disease progression and outcome including infecting strain(s) genetic composition, age of acqui-sition, host immune response, environment, and diet. Due to the complexity of H. pylori infection, all possible host-pathogen interactions should be considered. One important factor frequently underplayed is host dietary habits which may serve a more important role during and immediately following infection with H. pylori than previously thought. It has been shown that people consuming diets rich in fruits and vegetables have a lower incidence of H. pylori infection and severe gastric malignancies and that this may be in part due to natural bioactive compounds acting against H. pylori. We believe that alternative, diet-based strategies may have a significant impact on attenuating this bacte-rium when used alone or in combination with current antibiotic regimes; however, natural compounds should be better studied to understand their full therapeutic potential. Muscadine grapes, because of their high levels of polyphenolic compounds and unique chemical makeup, are believed to be a proper candidate for study since these fruits have already been shown to exert anti-H. pylori activity in vitro with effects possi-bly related to inhibition of bacterial attachment and virulence factors. The objectives of this study were to 1) identify active phenolic compounds in muscadine grape skin respon-sible for anti-H. pylori activity, 2) use DNA microarrays to study gene expression upon iii exposure to active compounds, and 3) explore the preventative potential of muscadine grape skin and identified polyphenols for ameliorating H. pylori infection and/or the H. pylori-induced inflammatory response in mice. Our results show that muscadine grape skin and major phenolics quercetin and resveratrol exert strong anti-H. pylori activity; especially when in combination, and may have the potential to be incorporated into an effective, diet-based approach for the pre-vention and/or treatment of H. pylori infection

    Innate immune defence to Helicobacter pylori.

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    Helicobacter pylori exhibits tropism for the human stomach causing a spectrum of complications ranging from gastritis to gastric cancer in susceptible individuals. The mechanism(s) that allow the bacteria to persist and cause disease are unfolding. p-defensins are a family of endogenous, epithelial anti-microbial peptides that engage in host defense most prominently at mucosal surfaces. We and others have previously shown that human p-defensin (hBD)-2 and -3 are potent bactericidal agents against H. pylori. At present the identity of signalling pathways involved in host-bacterial cross talk leading to modulation of host antimicrobial immunity are unknown. The present study firstly investigated the potential role of bacterial virulence factors in mediating human p-defensin gene expression during H. pylori infection. AGS gastric epithelial cells were infected with cytotoxic H. pylori strains (60190, 84-183) and isogenic mutant strains (cagA-, cagE-, vacA- and CagPAl-). Human p-defensin (hBD2 & -3) gene expression quantified by RT-PCR and p-defensin transcriptional regulation was followed by transient transfection studies utilising hBD2 and -3 promoter luciferase constructs. We found hBD2 induction was dependent upon an intact cagPAI and minimal involvement was observed for the bacterial virulence factors CagA and VacA in modulating P-defensin expression. We sought to investigate the bacterial component responsible for instigating epithelial innate immune responses. Through the use of siRNA for NODI we determined a role for NODI-dependent NF-kB activation in mediating hBD2 but not hBD3 expression. Experiments utilising specific inhibitors of the MAP Kinase pathways directed us to delineate the role of each pathway in modulating p-defensin expression by the activation of stably transfected conditional MAP Kinase mutants. These studies revealed critical involvement of ERK pathway in the regulation of hBD3 but not hBD2 gene expression. Signalling upstream of ERK was explored and revealed EGFR as the host receptor responsible for detection and initiation of hBD3 gene and peptide production. Our studies demonstrated a crucial role for NODI in H. /Ty/ort-mediated hBD2 but not hBD3 expression and implicate EGFR transactivation in mediating hBD3 but not hBD2 expression, thus indicating two distinct regulatory mechanisms at play during innate immune host response to H. pylori infection
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