Arnoud H. M. van Vliet

Pathogenesis of Helicobacter pylori Infection

SUMMARY Helicobacter pylori is the first formally recognized bacterial carcinogen and is one of the most successful human pathogens, as over half of the worlds population is colonized with this gram-negative bacterium. Unless treated, colonization usually persists lifelong. H. pylori infection represents a key factor in the etiology of various gastrointestinal diseases, ranging from chronic active gastritis without clinical symptoms to peptic ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Disease outcome is the result of the complex interplay between the host and the bacterium. Host immune gene polymorphisms and gastric acid secretion largely determine the bacteriums ability to colonize a specific gastric niche. Bacterial virulence factors such as the cytotoxin-associated gene pathogenicity island-encoded protein CagA and the vacuolating cytotoxin VacA aid in this colonization of the gastric mucosa and subsequently seem to modulate the hosts immune system. This review focuses on the microbiological, clinical, immunological, and biochemical aspects of the pathogenesis of H. pylori.

HELICOBACTER PYLORI AND ANTIMICROBIAL RESISTANCE: MOLECULAR MECHANISMS AND CLINICAL IMPLICATIONS

Helicobacter pylori is an important human pathogen that colonises the stomach of about half of the worlds population. The bacterium has now been accepted as the causative agent of several gastroduodenal disorders, ranging from chronic active gastritis and peptic ulcer disease to gastric cancer. The recognition of H pylori as a gastric pathogen has had a substantial effect on gastroenterological practice, since many untreatable gastroduodenal disorders with uncertain cause became curable infectious diseases. Treatment of H pylori infection results in ulcer healing and can reduce the risk of gastric cancer development. Although H pylori is susceptible to many antibiotics in vitro, only a few antibiotics can be used in vivo to cure the infection. The frequent indication for anti-H pylori therapy, together with the limited choice of antibiotics, has resulted in the development of antibiotic resistance in H pylori, which substantially impairs the treatment of H pylori-associated disorders. Antimicrobial resistance in H pylori is widespread, and although the prevalence of antimicrobial resistance shows regional variation per antibiotic, it can be as high as 95%. We focus on the treatment of H pylori infection and on the clinical relevance, mechanisms, and diagnosis of antimicrobial resistance.

Next generation sequencing of microbial transcriptomes: challenges and opportunities

Over the past 15 years, microbial functional genomics has been made possible by the combined power of genome sequencing and microarray technology. However, we are now approaching the technical limits of microarray technology, and microarrays are now being superseded by transcriptomics based on high-throughput (next generation) DNA-sequencing technologies. The term RNA-seq has been coined to represent transcriptomics by next-generation sequencing. Although pioneered on eukaryotic organisms due to the relative ease of working with eukaryotic mRNA, the RNA-seq technology is now being ported to microbial systems. This review will discuss the opportunities of RNA-seq transcriptome sequencing for microorganisms, and also aims to identify challenges and pitfalls of the use of this new technology in microorganisms.

The Complete Genome Sequence of Campylobacter jejuni Strain 81116 (NCTC11828)

Campylobacter jejuni is a major human enteric pathogen that displays genetic variability via genomic reorganization and phase variation. This variability can adversely affect the outcomes and reproducibility of experiments. C. jejuni strain 81116 (NCTC11828) has been suggested to be a genetically stable strain (G. Manning, B. Duim, T. Wassenaar, J. A. Wagenaar, A. Ridley, and D. G. Newell, Appl. Environ. Microbiol. 67:1185-1189, 2001), is amenable to genetic manipulation, and is infective for chickens. Here we report the finished annotated genome sequence of C. jejuni strain 81116.

The Helicobacter pylori Homologue of the Ferric Uptake Regulator Is Involved in Acid Resistance

ABSTRACT The only known niche of the human pathogen Helicobacter pylori is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylori genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella enterica serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pylori Fur homologue in acid resistance. Isogenic fur mutants were generated in three H. pylori strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the fur mutants was severely impaired. No differences were observed between the survival of the fur mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the fur mutant at acidic pH. This indicates that the phenotype of the fur mutant at low pH was not due to increased iron sensitivity. Transcription of fur was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.

Regulation of Ferritin-Mediated Cytoplasmic Iron Storage by the Ferric Uptake Regulator Homolog (Fur) of Helicobacter pylori

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.

Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level

ABSTRACT The nickel-containing enzyme urease is an essential colonization factor of the gastric pathogen Helicobacter pylori, as it allows the bacterium to survive the acidic conditions in the gastric mucosa. Although urease can represents up to 10% of the total protein content of H. pylori, expression of urease genes is thought to be constitutive. Here it is demonstrated that H. pyloriregulates the expression and activity of its urease enzyme as a function of the availability of the cofactor nickel. Supplementation of brucella growth medium with 1 or 100 μM NiCl2 resulted in up to 3.5-fold-increased expression of the urease subunit proteins UreA and UreB and up to 12-fold-increased urease enzyme activity. The induction was specific for nickel, since the addition of cadmium, cobalt, copper, iron, manganese, or zinc did not affect the expression of urease. Both Northern hybridization studies and a transcriptionalureA::lacZ fusion demonstrated that the observed nickel-responsive regulation of urease is mediated at the transcriptional level. Mutation of the HP1027 gene, encoding the ferric uptake regulator (Fur), did not affect the expression of urease in unsupplemented medium but reduced the nickel induction of urease expression to only twofold. This indicates that Fur is involved in the modulation of urease expression in response to nickel. These data demonstrate nickel-responsive regulation of H. pyloriurease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.

Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

ABSTRACT The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O2) than under microaerobic conditions (5% O2, 10% CO2), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.

NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori

ABSTRACT The important human pathogen Helicobacter pylori requires the abundant expression and activity of its urease enzyme for colonization of the gastric mucosa. The transcription, expression, and activity of H. pylori urease were previously demonstrated to be induced by nickel supplementation of growth media. Here it is demonstrated that the HP1338 protein, an ortholog of the Escherichia coli nickel regulatory protein NikR, mediates nickel-responsive induction of urease expression in H. pylori. Mutation of the HP1338 gene (nikR) of H. pylori strain 26695 resulted in significant growth inhibition of the nikR mutant in the presence of supplementation with NiCl2 at ≥100 μM, whereas the wild-type strain tolerated more than 10-fold-higher levels of NiCl2. Mutation of nikR did not affect urease subunit expression or urease enzyme activity in unsupplemented growth media. However, the nickel-induced increase in urease subunit expression and urease enzyme activity observed in wild-type H. pylori was absent in the H. pylori nikR mutant. A similar lack of nickel responsiveness was observed upon removal of a 19-bp palindromic sequence in the ureA promoter, as demonstrated by using a genomic ureA::lacZ reporter gene fusion. In conclusion, the H. pylori NikR protein and a 19-bp operator sequence in the ureA promoter are both essential for nickel-responsive induction of urease expression in H. pylori.

Iron-Responsive Regulation of the Helicobacter pylori Iron-Cofactored Superoxide Dismutase SodB Is Mediated by Fur

Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.