Article

Natural Competence Promotes Helicobacter pylori Chronic Infection

Division of Human Biology, Fred Hutchinson Cancer Research Center. 1100 Fairview Ave. N, Seattle, WA 98109.
Infection and immunity (Impact Factor: 3.73). 10/2012; 81(1). DOI: 10.1128/IAI.01042-12
Source: PubMed

ABSTRACT

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with
regard to organisms with restricted host ranges, such as the human stomach pathogen Helicobacter pylori. In most cases, H. pylori infects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute
infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2
months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection,
the cagY component of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both
bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population of cagY+ and cagY mutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in
addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common in H. pylori strains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane
DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was
normal. Thus, exchange of DNA between genetically heterogeneous H. pylori strains may improve chronic colonization. The strains and methods described here will be important tools for defining both
the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

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Available from: Ilana E Cohen, Jul 16, 2014
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    • "The analysis for " primer " selection consisted on: an optimal reaction efficiency and specific melting curves of the qPCR reaction. Also, as H. pylori is a pathogen that can survive to the hostile environment of the gastric tissue, there is low probability of contamination with other bacterial pathogens [6]. The primers selected for analyses were HP6 forward (5 0 -AGACACGGTCCAGACTCCTA-3 0 ) and reverse (5 0 -ACGCCCAGTGATTCCGAGTA-3 0 ) based on the best melting curve and reaction efficiency (Fig. 6). "
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    ABSTRACT: To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. • Use of at least 25 cut sections of 10–20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut. • Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position. • The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.
    Full-text · Article · Dec 2015 · MethodsX
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    ABSTRACT: The bacterial pathogen Helicobacter pylori has co-evolved with humans and colonizes approximately 50% of the human population, but only causes overt gastric disease in a subset of infected hosts. In this Review, we discuss the pathogenesis of H. pylori and the mechanisms it uses to promote persistent colonization of the gastric mucosa, with a focus on recent insights into the role of the virulence factors vacuolating cytotoxin (VacA), cytotoxin-associated gene A (CagA) and CagL. We also describe the immunobiology of H. pylori infection and highlight how this bacterium manipulates the innate and adaptive immune systems of the host to promote its own persistence.
    No preview · Article · May 2013 · Nature Reviews Microbiology
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    ABSTRACT: Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O2 (5% O2, 7.6% CO2, 7.6% H2) and high O2 (15% O2, 2.9% CO2, 2.9% H2) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O2 tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O2 concentration for these initial experiments also changed the concentrations of CO2 and H2, transformations were repeated under conditions where O2, CO2, and H2 were each varied individually. The results showed that the increase in transformation efficiency under high O2 was largely due to a decrease in CO2. An increase in pH similar to that caused by low CO2 was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.
    Preview · Article · Nov 2013 · Journal of bacteriology
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