Inactivation of the gene encoding the cationic antimicrobial peptide resistance factor MprF increases biofilm formation but reduces invasiveness of Listeria monocytogenes.

AIMS: To understand the genetics involved in surface attachment and biofilm formation of Listeria monocytogenes. METHODS AND RESULTS: An in vitro screen of a Himar1 transposon library of L. monocytogenes strain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild-type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild-type. The mutant 15G01 mprF::Himar1, which had a transposon insertion in the mprF gene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01 mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo-2 intestinal cells, suggesting virulence properties are compromised by the inactivation of mprF. CONCLUSIONS: Biofilm formation and gallidermin resistance of L. monocytogenes is influenced by mprF, but this trait is associated with a compromise in invasiveness. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of the mprF gene results in enhanced biofilm formation and abiotic surface attachment of L. monocytogenes.

Tetrazolium reduction allows assessment of biofilm formation by Campylobacter jejuni in a food matrix model.

AIMS: To develop a staining method for specific detection of metabolically active (viable) cells in biofilms of the foodborne pathogen Campylobacter jejuni. METHODS AND RESULTS: Conversion of 2,3,5 triphenyltetrazolium chloride (TTC) to insoluble, red 1,3,5-triphenylformazan (TPF) was dependent on metabolic activity of Camp. jejuni. When used with chicken juice, TTC staining allowed quantification of Camp. jejuni biofilm levels, whereas the commonly used dye, crystal violet, gave high levels of nonspecific staining of food matrix components (chicken juice). The assay was optimized to allow for monitoring of biofilm levels and adapted to monitor levels of Camp. jejuni in broth media. CONCLUSIONS: Staining with TTC allows for the quantification of metabolically active Camp. jejuni and thus allows for quantification of viable cells in biofilms and food matrices. The TTC staining method can be adapted to quantify bacterial cell concentration in a food matrix model, where the accepted method of A600 measurement is not suitable due to interference by components of the food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: 2,3,5 Triphenyltetrazolium chloride (TTC) staining is a low-cost technique suitable for use in biofilm analysis, allowing rapid and simple imaging of metabolically active cells and increasing the methods available for biofilm assessment and quantification.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Acid-shock of Campylobacter jejuni induces flagellar gene expression and host cell invasion.

The bacterial pathogen Campylobacter jejuni is the leading cause of foodborne gastroenteritis in the developed world, with the organism being transmitted by ingestion of contaminated and undercooked poultry. Exposure to acid is an inevitable stressor for C. jejuni during gastric passage, yet the effect of low pH on C. jejuni virulence is still poorly understood. Here, we investigate the effect of acid-shock on C. jejuni viability, gene expression and host-cell invasion. C. jejuni strain NCTC 11168 survived acid exposure at pH 3.5 and above for up to 30 min without a drop in viability, and this exposure induced the expression of flagellar genes transcribed from σ(54)-dependent promoters. Furthermore, acid-shock resulted in increased C. jejuni invasion of m-ICcl2 mouse small intestine crypt cells grown on transwells, but not when the cells were grown on flat-bottomed wells. This suggests that C. jejuni might be invading intestinal epithelial cells at the basolateral side, possibly after paracellular passage. We hypothesize that acid-shock prior to intestinal entry may serve as a signal that primes C. jejuni to express its virulence gene repertoire including flagellar motility genes, but this requires further study in the context of an appropriate colonization or disease model.

Campylobacter bacteremia: a rare and under-reported event?

Bacteria belonging to the species Campylobacter are the most common cause of bacterial diarrhoea in humans. The clinical phenotype associated with Campylobacter infections ranges from asymptomatic conditions to severe colitis and bacteremia. In susceptible patients, Campylobacter infections are associated with significant morbidity and mortality, with both host factors and bacterial factors being involved in the pathogenesis of bacteremia. In the host, age, gender and immune-compromising conditions may predispose for Campylobacter infections, whilst the most important bacterial determinants mentioned in the literature are cytotoxin production and flagellar motility. The role of sialylated lipo-oligosaccharide (LOS) and serum resistance in bacteremia is inconclusive at this time, and the clinical significance of Campylobacter bacteremia is not yet fully understood. More emphasis on the detection of Campylobacter species from blood cultures in susceptible patients at risk for Campylobacter infections will increase our understanding of the pathogenesis and the relevance of Campylobacter bacteremia.

PerR controls peroxide- and iron-responsive expression of oxidative stress defense genes in Helicobacter hepaticus.

Chronic intestinal and hepatic colonization with the microaerophilic murine pathogen Helicobacter hepaticus can lead to a range of inflammatory diseases of the lower digestive tract. Colonization is associated with an active cellular immune response and production of oxygen radicals. During colonization, H. hepaticus needs to cope with and respond to oxidative stress, and here we report on the role of the H. hepaticus PerR-regulator (HH0942) in the expression of the peroxidase-encoding katA (HH0043) and ahpC (HH1564) genes. Transcription of katA and ahpC was induced by hydrogen peroxide, and by iron restriction of growth media. This iron- and hydrogen peroxide-responsive regulation of katA and ahpC was mediated at the transcriptional level, from promoters directly upstream of the genes. Inactivation of the perR gene resulted in constitutive, iron-independent high-level expression of the katA and ahpC transcripts and corresponding proteins. Finally, inactivation of the katA gene resulted in increased sensitivity of H. hepaticus to hydrogen peroxide and reduced aerotolerance. In H. hepaticus, iron metabolism and oxidative stress defense are intimately connected via the PerR regulatory protein. This regulatory pattern resembles that observed in the enteric pathogen Campylobacter jejuni, but contrasts with the pattern observed in the closely related human gastric pathogen Helicobacter pylori.

Genomics of thermophilic campylobacter species.

The thermophilic Campylobacter species C. jejuni and C. coli are important human pathogens, which are major causes of bacterial gastroenteritis. The recent progress in genomics techniques has allowed for a rapid increase in our knowledge of the molecular biology of Campylobacter species, but needs to be matched by concurrent increases in our understanding of the unique biology of these organisms. Campylobacter species display significant levels of genomic variation via natural transformation, phase variation, plasmid transfer and infection with bacteriophages, and this poses a continuous challenge for studies on pathogenesis, physiology, epidemiology and evolution of Campylobacter. In this chapter we will review the current state of the art of the genomics of thermophilic Campylobacter species, and opportunities where genomics can further contribute to our understanding of the biology of these successful human pathogens.

A pro-inflammatory genotype predisposes to Barrett's esophagus.

INTRODUCTION: Severity of mucosal inflammation is shown to be associated with Barrett's esophagus (BE) development in animals. It has therefore been postulated that a strong pro-inflammatory host response predisposes to BE. AIM: To determine the impact of cytokine gene polymorphisms on the development of BE. METHODS: The multiplex SNaPshot method was used to determine interleukin (IL)-12B (A+1188C), IL-10 (C-592A, C-819T, A-1082G), IL-8 (A-251T), IL-6 (G-174C) and IL-2 (G-330T) gene polymorphisms in 255 patients with BE and 247 patients with reflux esophagitis (RE). RESULTS: The presence of the IL-12B C-allele, which is associated with increased IL-12p70 expression, was more frequently observed in BE than in RE patients [odds ratio (OR) 1.8; 95% confidence interval (CI) 1.2-2.7; P = 0.007). The risk of BE was increased in patients in whom the IL-12B C-allele coincided with a hiatal hernia (OR 2.9; 95% CI 1.32-6.58; P = 0.008). The IL-10(-1082) GG genotype, which is associated with higher IL-10 levels, was also associated with a decreased risk of BE when it was associated with the IL-12B C-allele, indicating IL-10-dependent down-regulation of IL-12p70 expression. A combination of the IL-12B AA genotype and the IL-10 AA or AG genotypes was associated with RE (OR 1.4; 95% CI 1.05-1.85; P = 0.011). CONCLUSION: A genetic profile predisposing to a strong pro-inflammatory host response, mediated by IL-12p70 and partially dependent on IL-10, is associated with BE. This risk further increases when this genotype coincides with a hiatal hernia, suggesting that exposure to gastroesophageal reflux in the presence of a pro-inflammatory genetic background is a driving force in the development of BE.

The homeodomain protein CDX2 is an early marker of Barrett's oesophagus.

BACKGROUND: In Barrett's oesophagus (BO), squamous epithelium is replaced by specialised intestinal epithelium (SIE). Transcription factors associated with intestinal differentiation, such as CDX2, may be involved in BO development. AIM: To investigate CDX2 expression in BO, squamous epithelium, and oesophageal adenocarcinoma (ADC). METHODS: CDX2 expression was assessed in 245 samples-167 biopsies of the columnar lined segment and 38 squamous epithelial biopsies of 39 patients with histologically confirmed BO (10 with ADC). Forty biopsies from 20 patients with reflux oesophagitis (RO) without BO were also evaluated. CDX2 protein was investigated immunohistochemically in 138 biopsies from 16 patients with BO, four with ADC, and 20 with RO. Cdx2 and Muc2 mRNA were detected semiquantitatively using 88 BO biopsies and squamous epithelium from 19 BO patients, and when present from ADC. RESULTS: SIE was present in 53/79 biopsies from the columnar lined segment; CDX2 protein was seen in all epithelial cells, but not in biopsies containing only gastric metaplastic epithelium (26/79), or in squamous epithelium (0/40) of patients with RO. Cdx2 mRNA was detected in all biopsies with goblet cell specific Muc2 transcription-indicative of SIE. Low Cdx2 mRNA expression was seen in 6/19 squamous epithelium samples taken 5 cm above the squamocolumnar junction of BO patients. CONCLUSION: CDX2 protein/mRNA is strongly associated with oesophageal SIE. Cdx2 mRNA was present in the normal appearing squamous epithelium of one third of BO patients, and may precede morphological changes seen in BO. Therefore, pathways that induce Cdx2 transcription in squamous epithelial cells may be important in BO development.

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

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. pylori regulates 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 microM NiCl(2) 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 transcriptional ureA::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. pylori urease, a phenomenon likely to be of importance during the colonization and persistence of H. pylori in the gastric mucosa.

The iron-induced ferredoxin FdxA of Campylobacter jejuni is involved in aerotolerance.

A gene encoding a putative 2[4Fe--4S] ferredoxin (FdxA) was identified upstream of, and divergent to the peroxide stress defense gene ahpC of the microaerophilic pathogen Campylobacter jejuni. The transcription start site of fdxA was located 27 and 28 bp upstream of the fdxA start codon. Transcriptional fusions of the fdxA promoter to a lacZ reporter gene demonstrated that expression of fdxA is iron-induced, and thus oppositely regulated to the iron-repressed ahpC gene. Insertional mutagenesis of the fdxA gene did not affect microaerobic growth of C. jejuni, but significantly reduced aerotolerance of C. jejuni. The fdxA gene is the first reported iron-induced gene of C. jejuni, and encodes a novel component of its oxidative stress defense.

The role of Helicobacter pylori virulence factors in interleukin production by monocytic cells.

Helicobacter pylori infection results in chronic gastritis, which is initiated by the release of cytokines like interleukin (IL)-12 and IL-8 from mononuclear cells, and IL-8 from gastric epithelial cells. The severity of gastritis is influenced both by host factors and by bacterial factors such as the Cag proteins and the vacuolating cytotoxin VacA. Amounts of IL-12 and IL-8 produced by monocytic THP-1 cells differed considerably between the eight H. pylori isolates tested, but in contrast to H. pylori-induced IL-8 production by gastric epithelial cells, did not correlate to the Cag and VacA types of the strains. Apparently, in addition to Cag and VacA, other bacterial factors determine the extent in which H. pylori induced IL production in monocytes.

Identification of environmental stress-regulated genes in Helicobacter pylori by a lacZ reporter gene fusion system.

BACKGROUND: Helicobacter pylori persists in the human stomach for decades. This requires an efficient adaptation of H. pylori to the gastric niche and involves the regulation of bacterial genes in response to environmental stress. Efficient molecular tools to identify regulated H. pylori genes are scarce, therefore we developed a genomic lacZ reporter gene fusion system in H. pylori to screen for stress-regulated genes. MATERIALS AND METHODS: The integration vector pBW was constructed and used to generate random genomic lacZ fusions in H. pylori. Two-hundred-and-fifty H. pylori transformants were selected from this library, replica-plated and screened for differential lacZ expression after exposure to two environmental stress conditions: increased temperature (42 degrees C), and iron-limitation. RESULTS: From a library of H. pylori transformants with random genomic transcriptional lacZ fusions, two stress-regulated H. pylori loci were identified. The transcription of a gene of unknown function (designated hsp12) was increased by incubation at 42 degrees C. The transcription of a locus, consisting of the three fumarate reductase subunit genes (frdCAB) and the HP0190 gene from H. pylori strain 26695, was decreased under iron-limitation. CONCLUSIONS: This is the first time that a genomic transcriptional lacZ reporter gene H. pylori library has been used as a tool for the fast and efficient identification of environmental stress-regulated H. pylori genes.

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.

The iron-responsive regulator Fur of Campylobacter jejuni is expressed from two separate promoters.

A lacZ-based reporter gene system was used to identify the promoter of the Campylobacter jejuni iron-responsive gene regulator Fur. In other Gram-negative bacteria, the fur promoter is usually located directly upstream of the fur gene and is often autoregulated in response to iron. In this study we demonstrate that expression of the C. jejuni fur gene is controlled from two promoters located in front of the first and second open reading frames upstream of fur. Neither of these promoters was iron-regulated, and the presence of both promoters in front of fur gives higher expression of the lacZ reporter than with either promoter alone. Expression from two distal promoters might be a mechanism for regulating the level of the C. jejuni Fur protein in response to unknown stimuli.

Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp).

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences.

Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.

Campylobacter jejuni contains two fur homologs: characterization of iron-responsive regulation of peroxide stress defense genes by the PerR repressor.

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.