Helicobacter pylori Genetic Polymorphisms in Gastric Disease Development.

Infecting half of the world's population, Helicobacter pylori is a medically important bacterium that induces a variety of gastric diseases, including gastritis, peptic ulcer disease, and gastric cancer. Sequencing of almost 1000 H. pylori isolates has revealed a diverse genome that contains abundant polymorphic genetic elements; many of these lie in factors likely to be associated with virulence. To ascertain the effect of these varying genetic elements, numerous epidemiological studies have investigated the contribution of the various polymorphisms to gastric disease development; particular focus has been placed on polymorphisms in the outer membrane proteins (OMPs), an effector protein, and a toxin produced by H. pylori. These studies have revealed geographic variation in the prevalence of various polymorphisms as well as in the associations between particular polymorphisms and gastric disease development. Furthermore, researchers have identified polymorphisms in multiple genes that frequently occur in combination. Though no single polymorphic genetic factor alone can fully account for gastric disease development in a population, the evaluation of multiple polymorphisms in a colonizing H. pylori strain can aid in the assessment of the pathogenic potential of the strain. Here we review specific H. pylori genetic polymorphisms (Bab proteins, Hom proteins, HopQ, SabA, SabB, OipA, IceA, VacA and CagA) that have been linked to disease outcome and discuss how geographic location and virulence factor polymorphisms together contribute to H. pylori-induced disease.

Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix.

The concept of Helicobacter pylori biofilm formation is relatively new. To help provide a foundation for future biofilm studies, we characterized the biofilm formation ability of a common H. pylori 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 H. pylori 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 H. pylori biofilm formation.IMPORTANCE The study of H. pylori 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 H. pylori biofilm formation, it is not yet understood how H. pylori 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.

Heterologous Pseudomonas aeruginosa O-antigen delivery using a Salmonella enterica serovar Typhimurium wecA mutant strain.

There is a broad interest in adapting live vaccine strains (LVS) for use as recombinant vaccines that can deliver heterologous antigens. The Salmonella enterica serovar Typhimurium SL1344 ΔwecA LVS contains a mutation in wecA that abrogates production of Enterobacterial common antigen. This ΔwecA strain is attenuated in vivo, persistently colonizes the host, and protects against both wild type and cross-Salmonella serovar lethal challenge in a murine model of salmonellosis. Given these characteristics, we hypothesized that the SL1344 ΔwecA strain could be used as a carrier for heterologous antigen expression. To test this hypothesis, SL1344 ΔwecA was engineered to express the Pseudomonas aeruginosa O11 O-antigen gene cluster. Intraperitoneal (IP) but not oral immunization of BALB/c mice with the heterologous expression strain protected against lethal P. aeruginosa intranasal (IN) challenge. Furthermore, IP immunization resulted in P. aeruginosa O11-specific Ig and IgG antibody production. Functional analysis of sera collected from the IP immunized mice showed antibody-mediated agglutination and opsonophagocytic activity against P. aeruginosa. En masse, these results indicate that the S. Typhimurium SL1344 ΔwecA strain expressing the P. aeruginosa O11 O-antigen gene cluster is able to induce a humoral immune response and to protect against lethal P. aeruginosa challenge. As such, the S. Typhimurium SL1344 ΔwecA LVS can likely serve as a vehicle for expression of a wide variety of heterologous antigens as a means to create recombinant vaccines.

An enterobacterial common antigen mutant of Salmonella enterica serovar Typhimurium as a vaccine candidate.

Due to increasing rates of invasive Salmonella enterica serovar Typhimurium infection, there is a need for an effective vaccine to prevent this disease. Previous studies showed that a mutation in the first gene of the Enterobacterial common antigen biosynthetic pathway, wecA, resulted in attenuation of S. Typhimurium in a murine model of salmonellosis. Furthermore, immunization with a wecA(-) strain protected against lethal challenge with the parental wild type S. Typhimurium strain. Herein, we examined whether the S. Typhimurium wecA(-) strain could also provide cross-protection against non-parental strains of S. Typhimurium and S. Enteritidis. We found that intraperitoneal immunization (IP) with S. Typhimurium SL1344 wecA(-) resulted in a significant increase in survival compared to control mice for all Salmonella challenge strains tested. Oral immunization with SL1344 wecA(-) also resulted in increased survival; however, protection was less significant than with intraperitoneal immunization. The increase in survival of SL1344 wecA(-) immunized mice was associated with a Salmonella-specific IgG antibody response. Furthermore, analysis of sera from IP and orally immunized animals revealed cross-reactive antibodies to numerous Salmonella isolates. Functional analysis of antibodies found within the sera from IP immunized animals revealed agglutination and opsonophagocytic activity against all tested O:4 Salmonella serovars. Together these results indicate that immunization with a S. Typhimurium wecA(-) strain confers protection against lethal challenge with wild type S. Typhimurium and S. Enteritidis and that immunization correlates with functional antibody production.

Helicobacter pylori attachment-blocking antibodies protect against duodenal ulcer disease.

The majority of the world population carry the gastric pathogen Helicobacter pylori. Fortunately, most individuals experience only low-grade or no symptoms, but in many cases the chronic inflammatory infection develops into severe gastric disease, including duodenal ulcer disease and gastric cancer. Here we report on a protective mechanism where H. pylori attachment and accompanying chronic mucosal inflammation can be reduced by antibodies that are present in a vast majority of H. pylori carriers. These antibodies block binding of the H. pylori attachment protein BabA by mimicking BabA's binding to the ABO blood group glycans in the gastric mucosa. However, many individuals demonstrate low titers of BabA blocking antibodies, which is associated with an increased risk for duodenal ulceration, suggesting a role for these antibodies in preventing gastric disease.

Nasal microbiota evolution within the congregate setting imposed by military training.

The human microbiome is comprised of a complex and diverse community of organisms that is subject to dynamic changes over time. As such, cross-sectional studies of the microbiome provide a multitude of information for a specific body site at a particular time, but they fail to account for temporal changes in microbial constituents resulting from various factors. To address this shortcoming, longitudinal research studies of the human microbiome investigate the influence of various factors on the microbiome of individuals within a group or community setting. These studies are vital to address the effects of host and/or environmental factors on microbiome composition as well as the potential contribution of microbiome members during the course of an infection. The relationship between microbial constituents and disease development has been previously explored for skin and soft tissue infections (SSTIs) within congregate military trainees. Accordingly, approximately 25% of the population carries Staphylococcus aureus within their nasal cavity, and these colonized individuals are known to be at increased risk for SSTIs. To examine the evolution of the nasal microbiota of U.S. Army Infantry trainees, individuals were sampled longitudinally from their arrival at Fort Benning, Georgia, until completion of their training 90 days later. These samples were then processed to determine S. aureus colonization status and to profile the nasal microbiota using 16S rRNA gene-based methods. Microbiota stability differed dramatically among the individual trainees; some subjects exhibited great stability, some subjects showed gradual temporal changes and some subjects displayed a dramatic shift in nasal microbiota composition. Further analysis utilizing the available trainee metadata suggests that the major drivers of nasal microbiota stability may be S. aureus colonization status and geographic origin of the trainees. Nasal microbiota evolution within the congregate setting imposed by military training is a complex process that appears to be affected by numerous factors. This finding may indicate that future campaigns to prevent S. aureus colonization and future SSTIs among high-risk military trainees may require a 'personalized' approach.

Generation of Isogenic Mutant Strains of Helicobacter pylori.

One important tool in the study of gene function is the construction of mutant strains. Specifically, the construction of isogenic mutant strains imparts researchers with the ability to compare a wild-type strain to a strain that is genetically identical with the exception of the gene of interest. For a bacterial species such as Helicobacter pylori , which is notorious for the genetic heterogeneity seen across isolates, comparisons between isogenic and parental strains control for the genetic variation seen between distinct isolates. This chapter details the construction of a clean gene deletion in which the entire coding region is replaced with a selectable marker. The approach detailed herein allows for the thorough investigation of gene function in the absence of confounding genetic variability.

Polymorphism in the CagA EPIYA motif impacts development of gastric cancer.

Helicobacter pylori causes diseases ranging from gastritis to peptic ulcer disease to gastric cancer. Geographically, areas with high incidences of H. pylori infection often overlap with areas with high incidences of gastric cancer, which remains one of the leading causes of cancer-related deaths worldwide. Strains of H. pylori that carry the virulence factor cytotoxin-associated gene A (cagA) are much more likely to be associated with the development of gastric cancer. Moreover, particular C-terminal polymorphisms in CagA vary by geography and have been suggested to influence disease development. We conducted a large-scale molecular epidemiologic analysis of South Korean strains and herein report a statistical link between the East Asian CagA EPIYA-ABD genotype and the development of gastric cancer. Characterization of a subset of the Korean isolates showed that all strains from cancer patients expressed and delivered phosphorylatable CagA to host cells, whereas the presence of the cagA gene did not strictly correlate to expression and delivery of CagA in all noncancer strains.

Balancing the double-edged sword: metal ion homeostasis and the ulcer bug.

The essential nature of many metals is counterbalanced by the toxic effect that they can exert on both the eukaryotic and prokaryotic cell when not properly controlled. As such, virtually all organisms have developed regulatory systems that are required to maintain metal ion homeostasis. Helicobacter pylori is arguably the most successful bacterial pathogen in the world; the bacterium colonizes more than 50% of the world's population. H. pylori lives in the acidic environment of the stomach and causes a persistent infection that results in disease sequelae such as gastritis, iron-deficiency anemia, ulcer disease and gastric cancer. A requirement of colonization is that the bacterium successfully competes with host cells for available metal ions. As such, it is perhaps no surprise that several crucial colonization factors utilize metal as an essential cofactor. Recent investigations into the absolute requirement for different metal ions and the need to manage their use have shown that metal ion homeostasis is achieved by H. pylori through the utilization of an intricate regulatory cascade that ensures metal uptake without toxic side effects. Herein we discuss this cascade, the role that individual metal ions play in H. pylori colonization and disease and the possibility that these metal homeostasis cascade components may serve as good targets for rational drug design to eradicate H. pylori infection.

Expanding the Helicobacter pylori genetic toolbox: modification of an endogenous plasmid for use as a transcriptional reporter and complementation vector.

Helicobacter pylori is an important human pathogen. However, the study of this organism is often limited by a relative shortage of genetic tools. In an effort to expand the methods available for genetic study, an endogenous H. pylori plasmid was modified for use as a transcriptional reporter and as a complementation vector. This was accomplished by addition of an Escherichia coli origin of replication, a kanamycin resistance cassette, a promoterless gfpmut3 gene, and a functional multiple cloning site to form pTM117. The promoters of amiE and pfr, two well-characterized Fur-regulated promoters, were fused to the promoterless gfpmut3, and green fluorescent protein (GFP) expression of the fusions in wild-type and delta fur strains was analyzed by flow cytometry under iron-replete and iron-depleted conditions. GFP expression was altered as expected based on current knowledge of Fur regulation of these promoters. RNase protection assays were used to determine the ability of this plasmid to serve as a complementation vector by analyzing amiE, pfr, and fur expression in wild-type and delta fur strains carrying a wild-type copy of fur on the plasmid. Proper regulation of these genes was restored in the delta fur background under high- and low-iron conditions, signifying complementation of both iron-bound and apo Fur regulation. These studies show the potential of pTM117 as a molecular tool for genetic analysis of H. pylori.

Use of Random and Site-Directed Mutagenesis to Probe Protein Structure-Function Relationships: Applied Techniques in the Study of Helicobacter pylori.

Mutagenesis is a valuable tool to examine the structure-function relationships of bacterial proteins. As such, a wide variety of mutagenesis techniques and strategies have been developed. This chapter details a selection of random mutagenesis methods and site-directed mutagenesis procedures that can be applied to an array of bacterial species. Additionally, the direct application of the techniques to study the Helicobacter pylori Ferric Uptake Regulator (Fur) protein is described. The varied approaches illustrated herein allow the robust investigation of the structural-functional relationships within a protein of interest.

A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro.

L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

Antibiotic treatment decreases microbial burden associated with pseudomyxoma peritonei and affects ß-catenin distribution.

PURPOSE: Pseudomyxoma peritonei is an understudied cancer in which an appendiceal neoplasm invades the peritoneum and forms tumor foci on abdominal organs. Previous studies have shown that bacteria reside within pseudomyxoma peritonei tumors and mucin. Thus, we sought to analyze the effect of antibiotics on bacterial density and ß-catenin expression within pseudomyxoma peritonei samples. EXPERIMENTAL DESIGN: The study included 48 patients: 19 with disseminated peritoneal adenomucinosis (DPAM) and 29 with peritoneal mucinous carcinomatosis (PMCA). Fourteen patients were given antibiotics (30 mg lansoprazole, 1 g amoxicillin, and 500 mg clarithromycin) twice a day for 14 days. One week after completion of therapy, surgery was conducted and specimens were harvested for pathology, bacterial culture, ISH, and immunohistochemistry. RESULTS: ISH showed the presence of bacteria in 83% of the patient samples, with a higher Helicobacter pylori density observed in PMCA versus DPAM. PMCA patients treated with antibiotics had a significantly lower bacterial density and decreased ß-catenin levels in the cytoplasm, the cell nuclei, and mucin-associated cells. Although not significant, similar trends were observed in DPAM patients. Cell membrane ß-catenin was significantly increased in both DPAM and PMCA patients receiving antibiotics. CONCLUSIONS: Bacteria play an important role in pseudomyxoma peritonei. Antibiotic treatment improved the histopathology of tissue, particularly in PMCA patients. In PMCA, antibiotics decreased bacterial density and were associated with a significant ß-catenin decrease in the cytoplasm, cell nuclei, and mucin along with a small membrane increase. These results suggest that antibiotics offer potential protection against cell detachment, cellular invasion, and metastasis.

Successful culture techniques for Helicobacter species: general culture techniques for Helicobacter pylori.

Half of the world's population is persistently infected with Helicobacter pylori. The chronicity of this infection ultimately elicits clinical manifestations ranging from gastritis and peptic ulcers to adenocarcinoma and MALT lymphoma. Laboratory research following the initial observations of Helicobacter species was greatly hindered by an inability to isolate and culture the bacteria. Thus, the ability to culture bacterial species from this genus is an extremely important step in expanding clinical knowledge and development of therapies. This chapter describes successful techniques for culturing H. pylori on selective horse blood agar media and in Brucella broth liquid media. Additionally, the specific growth requirements of other Helicobacter species are noted.

Successful culture techniques for Helicobacter species: establishing H. pylori cultures from infected rodents.

This chapter describes protocols for establishing H. pylori cultures from infected rodents.

Successful culture techniques for Helicobacter species: verification of Helicobacter identity using 16S rRNA gene sequence analysis.

This chapter describes protocols for the verification of putative Helicobacter species' identities using 16S rRNA gene sequence analysis.

A Tale of Two Toxins: Helicobacter Pylori CagA and VacA Modulate Host Pathways that Impact Disease.

Helicobacter pylori is a pathogenic bacterium that colonizes more than 50% of the world's population, which leads to a tremendous medical burden. H. pylori infection is associated with such varied diseases as gastritis, peptic ulcers, and two forms of gastric cancer: gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. This association represents a novel paradigm for cancer development; H. pylori is currently the only bacterium to be recognized as a carcinogen. Therefore, a significant amount of research has been conducted to identify the bacterial factors and the deregulated host cell pathways that are responsible for the progression to more severe disease states. Two of the virulence factors that have been implicated in this process are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which are cytotoxins that are injected and secreted by H. pylori, respectively. Both of these virulence factors are polymorphic and affect a multitude of host cellular pathways. These combined facts could easily contribute to differences in disease severity across the population as various CagA and VacA alleles differentially target some pathways. Herein we highlight the diverse types of cellular pathways and processes targeted by these important toxins.

A single nucleotide change affects fur-dependent regulation of sodB in H. pylori.

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter.