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  • 1
    Language: English
    In: PloS one, 2017, Vol.12(4), pp.e0176468
    Description: [This corrects the article DOI: 10.1371/journal.pone.0137078.].
    Keywords: Clinical Isolates ; Genotypes ; Genotypes ; Duodenal Ulcers ; Gastric Ulcers ; Gastritis ; Phylogenetic Analysis ; Red Blood Cells ; Statistical Distributions ; Variant Genotypes ; Helicobacter Pylori;
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PloS one, 2017, Vol.12(8), pp.e0183260
    Description: The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.
    Keywords: Bacterial Proteins -- Chemistry ; Helicobacter Pylori -- Enzymology ; Hydrogenase -- Chemistry
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: PloS one, 2015, Vol.10(8), pp.e0137078
    Description: Helicobacter pylori genetic variation is a crucial component of colonization and persistence within the inhospitable niche of the gastric mucosa. As such, numerous H. pylori genes have been shown to vary in terms of presence and genomic location within this pathogen. Among the variable factors, the Bab family of outer membrane proteins (OMPs) has been shown to differ within subsets of strains. To better understand genetic variation among the bab genes and to determine whether this variation differed among isolates obtained from different geographic locations, we characterized the distribution of the Bab family members in 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). Overall, we identified 23 different bab genotypes (19 in AH and 11 in KH), but only 5 occurred in greater than 5 isolates. Regardless of strain origin, a strain in which locus A and locus B were both occupied by a bab gene was the most common (85%); locus C was only occupied in those isolates that carried bab paralog at locus A and B. While the babA/babB/- genotype predominated in the KH (78.8%), no single genotype could account for greater than 40% in the AH collection. In addition to basic genotyping, we also identified associations between bab genotype and well known virulence factors cagA and vacA. Specifically, significant associations between babA at locus A and the cagA EPIYA-ABD motif (P〈0.0001) and the vacA s1/i1/m1 allele (P〈0.0001) were identified. Log-linear modeling further revealed a three-way association between bab carried at locus A, vacA, and number of OMPs from the HOM family (P〈0.002). En masse this study provides a detailed characterization of the bab genotypes from two distinct populations. Our analysis suggests greater variability in the AH, perhaps due to adaptation to a more diverse host population. Furthermore, when considering the presence or absence of both the bab and homA/B paralogs at their given loci and the vacA genotype, an association was observed. Our results highlight the multifactorial nature of H. pylori mediated disease and the importance of considering how the specific combinations of H. pylori virulence genes and their multiple interactions with the host will collectively impact disease progression.
    Keywords: Genotype ; Sequence Homology, Nucleic Acid ; Bacterial Outer Membrane Proteins -- Genetics ; Helicobacter Pylori -- Genetics
    E-ISSN: 1932-6203
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  • 4
    In: Helicobacter, April 2018, Vol.23(2), pp.n/a-n/a
    Description: To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.1111/hel.12461/abstract Byline: Stephanie L. Servetas, Aeryun Kim, Hanfu Su, Jeong-Heon Cha,D. Scott Merrell Keywords: Helicobacter pylori ; Hom; outer membrane proteins Abstract Background Helicobacter pylori encodes numerous outer membrane proteins (OMPs), but only a few have been characterized in depth. Deletion, duplication, and allelic variation of many of the H. pylori OMPs have been reported, which suggests that these proteins may play key roles in host adaptation. Herein, we characterize the variation observed within the Hom family of OMPs in H. pylori obtained from two geographically distinct populations. Materials and Methods PCR genotyping of the hom genes was carried out using clinical isolates from South Korea and the United States. A combination of statistical, phylogenetic, and protein modeling analyses was conducted to further characterize the hom variants. Results Variations in the closely related hom genes, homA and homB, occur in regions that are predicted to encode environmentally exposed loops. A similar phenomenon is true for homC.sub.S as compared to homC.sub.L. Conversely, little variation was observed in homD. Certain variants of the Hom family of proteins were more prominent in isolates from the Korean population as compared to isolates from the United States. Conclusion En masse, our data show that the homA, homB, and homC profiles vary based upon the geographic origin of the strain; however, the fourth member of the hom family, homD, is more highly conserved. Additionally, protein topology modeling showed that many of the less well-conserved regions between homA and homB and between homC.sub.S and homC.sub.L corresponded to predicted environmentally exposed loops, suggesting that the divergence of the Hom family may be due to host adaptation/pressure. Article Note: Stephanie L. Servetas and Aeryun Kim contributed equally to this work. CAPTION(S):
    Keywords: Helicobacter Pylori ; Hom ; Outer Membrane Proteins
    ISSN: 1083-4389
    E-ISSN: 1523-5378
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  • 5
    Language: English
    In: 한국미생물학회 학술대회논문집, 2017, Vol.2017(4), pp.255-255
    Source: DBpia - 디비피아 (Nurimedia)
    Source: DBpia (Nurimedia)
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  • 6
    Language: English
    In: Frontiers in microbiology, 2018, Vol.9, pp.1497
    Description: One elusive area in the field is an understanding of why some infections result in gastric cancer, yet others persist asymptomatically for the life-span of the individual. Even before the genomic era, the high level of intraspecies diversity of was well recognized and became an intriguing area of investigation with respect to disease progression. Of interest in this regard is the unique repertoire of over 60 outer membrane proteins (OMPs), several of which have been associated with disease outcome. Of these OMPs, the association between HomB and disease outcome varies based on the population being studied. While the molecular roles for some of the disease-associated OMPs have been evaluated, little is known about the role that HomB plays in the lifecycle. Thus, herein we investigated expression, regulation, and contribution to biofilm formation. We found that in strain G27, was expressed at a relatively low level until stationary phase. Furthermore, expression was suppressed at low pH in an ArsRS-dependent manner; mutation of resulted in increased transcript at all tested time-points. ArsRS regulation of appeared to be direct as purified ArsR was able to specifically bind to the promoter. This regulation, combined with our previous finding that ArsRS mutations lead to enhanced biofilm formation, led us to test the hypothesis that contributes to biofilm formation by . Indeed, subsequent biofilm analysis using a crystal-violet quantification assay and scanning electron microscopy (SEM) revealed that loss of from hyper-biofilm forming strains resulted in reversion to a biofilm phenotype that mimicked wild-type. Furthermore, expression of from a promoter that negated ArsRS regulation led to enhanced biofilm formation even in strains in which the chromosomal copy of had been deleted. Thus, is necessary for hyper-biofilm formation of ArsRS mutant strains and aberrant regulation of this gene is sufficient to induce a hyper-biofilm phenotype. In summary, these data suggest that the ArsRS-dependent regulation of OMPs such as HomB may be one mechanism by which ArsRS dictates biofilm development in a pH responsive manner.
    Keywords: Arsrs ; H. Pylori ; Omps ; Biofilms ; Homb
    ISSN: 1664-302X
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  • 7
    Language: English
    In: Frontiers in microbiology, 2015, Vol.6, pp.558
    Description: Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at neutral pH has been demonstrated.
    Keywords: Helicobacter ; Arsrs ; Nikr ; Ph ; Pylori ; Regulation ; Urease
    ISSN: 1664-302X
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  • 8
    Language: English
    In: BMC genomics, 04 February 2014, Vol.15, pp.96
    Description: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.
    Keywords: High-Throughput Nucleotide Sequencing -- Instrumentation
    E-ISSN: 1471-2164
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  • 9
    Language: English
    In: Journal of Bacteriology, 2016, Vol.198(17-18), p.2536(13)
    Description: Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ?nikR ?arsS, and ?fur ?nikR ?arsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation.
    Keywords: Helicobacter Pylori – Research ; Helicobacter Pylori – Physiological Aspects ; Microbial Mats – Research
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 10
    Language: English
    In: Applied and environmental microbiology, 15 July 2018, Vol.84(14)
    Description: The concept of biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common lab strain, G27. The goal of this study was to evaluate biofilm formation by G27 in response to common culture conditions and to explore the biofilm matrix. Our results indicate that while various types of growth media did not dramatically affect biofilm formation, surface selection had a significant effect on the final biofilm mass. Furthermore, enzymatic assays and confocal microscopy revealed that proteins appear to be the primary structural component of the extracellular matrix; extracellular DNA (eDNA) and polysaccharides were also present but appear to play a secondary role. Finally, we found that two well-characterized antibiofilm cationic peptides differentially affected early and late-stage biofilms. Together these results provide interesting avenues for future investigations that will seek to understand biofilm formation. The study of biofilm formation is still in its infancy. As such, there is great variability in how biofilm assays are performed across labs. While several groups have begun to investigate factors that influence biofilm formation, it is not yet understood how biofilm formation may vary based on commonly used conditions. These inconsistencies lead to difficulties in interpretation and comparison between studies. Here, we set out to characterize biofilm formation by a commonly available lab strain, G27. Our findings provide novel insight into optimal biofilm conditions, the biofilm matrix, and possible mechanisms to block or disrupt biofilm formation.
    Keywords: Helicobacter Pylori ; Antimicrobial Peptides ; Biofilms ; Biofilms -- Growth & Development ; DNA, Bacterial -- Isolation & Purification ; Extracellular Polymeric Substance Matrix -- Metabolism ; Helicobacter Pylori -- Growth & Development
    ISSN: 00992240
    E-ISSN: 1098-5336
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