HtrA-dependent adherence and invasion of Campylobacter jejuni in human vs avian cells.

The aim of this study was to investigate whether HtrA is responsible for differences in adherence and invasion of Campylobacter jejuni towards human and chicken cell lines. Gentamicin protection assays were performed with either human Caco-2 or chicken 2G4 cells using C. jejuni strain NCTC11168 to compare the adhesion and invasion rates towards these two cell types. The results revealed significant differences in the adhesion and invasion rates between the human and avian cells. Deletion of the Campylobacter htrA gene, coding for the dual function of serine protease and chaperonin with a role in pathogenesis, led to a reduction of the rates in both cell lines. Using a single-amino acid substitution mutant (ΔhtrA/htrAS197A ) that lacked protease activity, but retained chaperonin activity, we show that the first is involved in the invasion of human Caco-2 and chicken 2G4 cells, whereas the latter mutant invaded at lower levels. Adherence towards the chicken cells is higher than towards Caco-2 cells and this is also dependent on HtrA. Together, these data suggest that the proteolytic activity of HtrA is involved in the difference in host response of C. jejuni towards human and chicken-derived cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter jejuni is the main cause for bacterial foodborne enterocolitis worldwide. While colonization of the human intestine can lead to severe problems, avian hosts - as the major source of infection - remain unaffected by the bacteria. We showed that the bacterial serine protease and chaperonin HtrA are involved in adhesion and invasion in both species and not responsible for the discrepancy of virulence between the different hosts. In future, HtrA might act as a target for inhibitors to avoid or eradicate colonization in chickens as a less problematic alternative to antibiotics in commercial livestock breeding.

Helicobacter pylori downregulates expression of human ß-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion.

Helicobacter pylori establishes a chronic lifelong infection in the human gastric mucosa, which may lead to peptic ulcer disease or gastric adenocarcinoma. The human beta-defensins (hßDs) are antimicrobial peptides, hßD1 being constitutively expressed in the human stomach. We hypothesized that H. pylori may persist, in part, by downregulating gastric hßD1 expression. We measured hßD1 and hßD2 expression in vivo in relation to the presence, density and severity of H. pylori infection, investigated differential effects of H. pylori virulence factors, and studied underlying signalling mechanisms in vitro. Significantly lower hßD1 and higher hßD2 mRNA and protein concentrations were present in gastric biopsies from infected patients. Those patients with higher-level bacterial colonization and inflammation had significantly lower hßD1 expression, but there were no differences in hßD2. H. pylori infection of human gastric epithelial cell lines also downregulated hßD1. Using wild-type strains and isogenic mutants, we showed that a functional cag pathogenicity island-encoded type IV secretion system induced this downregulation. Treatment with chemical inhibitors or siRNA revealed that H. pylori usurped NF-κB signalling to modulate hßD1 expression. These data indicate that H. pylori downregulates hßD1 expression via NF-κB signalling, and suggest that this may promote bacterial survival and persistence in the gastric niche.

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico.

AIMS: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. METHODS AND RESULTS: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 microg ml(-1)) and trimethoprim (MIC of >32 microg ml(-1)); few were resistant to clindamycin (MIC(90) 4 microg ml(-1)), erythromycin (MIC(90) 8 microg ml(-1)) and tetracycline (MIC(90) 8 microg ml(-1)); but none was resistant to azithromycin (MIC(90) 4 microg ml(-1)), ciprofloxacin (MIC(90) 1 microg ml(-1)) or gentamycin (MIC(90) 4 microg ml(-1)). Most strains restricted with SmaI, but a combination of SmaI-KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST-2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco-2 cells, medium cytotoxicity with INT-407 and Hep-2 cells and high cytotoxicity with CHO cells. CONCLUSION: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.

Rolling-circle replication of mitochondrial DNA in the higher plant Chenopodium album (L.).

The mitochondrial genomes of higher plants are larger and more complex than those of all other groups of organisms. We have studied the in vivo replication of chromosomal and plasmid mitochondrial DNAs prepared from a suspension culture and whole plants of the dicotyledonous higher plant Chenopodium album (L.). Electron microscopic studies revealed sigma-shaped, linear, and open circular molecules (subgenomic circles) of variable size as well as a minicircular plasmid of 1.3 kb (mp1). The distribution of single-stranded mitochondrial DNA in the sigma structures and the detection of entirely single-stranded molecules indicate a rolling-circle type of replication of plasmid mp1 and subgenomic circles. About half of the sigma-like molecules had tails exceeding the lengths of the corresponding circle, suggesting the formation of concatemers. Two replication origins (nicking sites) could be identified on mpl by electron microscopy and by a new approach based on the mapping of restriction fragments representing the identical 5' ends of the tails of sigma-like molecules. These data provide, for the first time, evidence for a rolling-circle mode of replication in the mitochondria of higher plants.

Evaluation of different plate media for direct cultivation of Campylobacter species from live broilers.

Accurate identification and optimal culturing procedures for Campylobacter spp. from live broilers are needed for epidemiological studies. Because there is no standardized protocol, we designed and conducted studies to evaluate different selective media for the culturing and isolation of Campylobacter spp. from cecal and fecal samples obtained from battery-reared and commercial broilers. Five media selective for Campylobacter were evaluated: Campylobacter agar base, Campylobacter, Campy-Line, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar. With contaminated broilers reared in battery cages, Campylobacter agar base, Campylobacter, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar revealed similar isolation rates (P > 0.05), whereas Campy-Line showed a lower efficacy (P < 0.05). With commercial live broilers, modified Campy-Cefex agar was more consistent for the isolation of Campylobacter from feces, whereas modified Campy-Cefex and modified charcoal cefoperazone deoxycholate agar showed similar isolation rates from cecal samples. Campy-Line agar showed a lower identification rate (P < 0.05) for both fecal and cecal samples. A multiplex PCR assay used for identification showed that Campylobacter jejuni and Campylobacter coli DNA was present in the samples. Pulsed field gel electrophoresis restriction profiles differed among samples collected from different commercial farms but were similar for isolates from the same farm, suggesting clonal differences. No variation was seen in pulsed field gel electrophoresis patterns among isolates cultured on different media. Our data suggest that the choice of plate medium may influence the efficiency of isolating Campylobacter spp. from broiler chickens by direct plating from fecal or cecal samples.

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We developed a fast, reliable PCR assay that specifically amplifies a 554-bp segment of the 16S rDNA from C. fetus. Fifty-two ATCC reference strains and 255 bacterial field isolates comprising the genera Campylobacter, Arcobacter, Helicobacter, Escherichia, Listeria, Salmonella, and Wolinella were evaluated using this PCR protocol. Only C. fetus strains were amplified. Sequence analysis of amplicons from ATCC and field strains of C. fetus confirmed the presence of the target DNA fragment. The detection limit of the technique was 5.9 x 10(3) CFU/ml. This PCR assay can yield reliable detection of C. fetus within 3 h after isolation of presumptive colonies on agar plates.

Quorum sensing dependent phenotypes and their molecular mechanisms in Campylobacterales.

Quorum sensing comprises the mechanism of communication between numerous bacteria via small signalling molecules, termed autoinducers (AI). Using quorum sensing, bacteria can regulate the expression of multiple genes involved in virulence, toxin production, motility, chemotaxis and biofilm formation, thus contributing to adaptation as well as colonisation. The current understanding of the role of quorum sensing in the lifecycle of Campylobacterales is still incomplete. Campylobacterales belong to the class of Epsilonproteobacteria representing a physiologically and ecologically diverse group of bacteria that are rather distinct from the more commonly studied Proteobacteria, such as Escherichia and Salmonella. This review summarises the recent knowledge on distribution and production of AI molecules, as well as possible quorum sensing dependent regulation in the mostly investigated species within the Campylobacterales group: Campylobacter jejuni and Helicobacter pylori.

Prevalence and spread of enterohepatic Helicobacter species in mice reared in a specific-pathogen-free animal facility.

Infections with enterohepatic Helicobacter species (EHS) can change the results of animal experiments. However, there is little information about the prevalence of EHS in noncommercial animal facilities. The aim of this study was to investigate the prevalence and the spread of EHS in specific-pathogen-free (SPF) mice. Fecal samples of 40 mouse lines were analyzed for members of the family Helicobacteraceae using a group-specific PCR targeting the 16S rRNA gene. Additional experiments were carried out to evaluate the spread of EHS among mice harbored in different caging systems. Helicobacter species were detected in 87.5% of the mouse lines tested. Five different Helicobacter species were identified: H. ganmani, H. hepaticus, H. typhlonicus, and the putative Helicobacter species represented by the isolates hamster B and MIT 98-5357. Helicobacter infection did not spread between animals in neighboring cages when individually ventilated cages were used; in contrast, when the mice were reared in open-air cages, EHS were found to spread from cage to cage. However, the spread was prevented by adding polycarbonate filter tops to the cages. When Helicobacter-negative and infected mice shared the same cage, transmission of the infection occurred in 100% within 2 weeks. Furthermore, we found that mice from commercial breeding facilities may carry undetected Helicobacter infections. Taken together, we show that infection with EHS may frequently occur and spread easily in mice reared under SPF conditions despite extensive safety precautions. Moreover, there is a high prevalence of rather uncommon Helicobacter species that may be a consequence of the current routine procedures used for health screening of SPF mice.

Strand switching during rolling circle replication of plasmid-like DNA circles in the mitochondria of the higher plant Chenopodium album (L.).

The structure of sigma-like mitochondrial DNA molecules prepared from suspension cultured cells of Chenopodium album (L.) was studied by electron microscopy. These molecules were highly variable in size, ranging from about 1 to 104 kb, and had single- and double-stranded regions typical for rolling circle replicating intermediates. Partial denaturation studies confirmed that these structures constitute rolling circles. Close inspection of the circle-tail junctions of the replication fork at high magnification suggests that in circles with a double-stranded tail, both strands of the tail seem to be covalently attached to the circle in about 27% of the molecules. This observation can be explained by a phenomenon called strand switching or strand splippage during rolling circle replication, similar to a mechanism proposed for bacterial replicons or in vitro replicating constructs harboring bacteriophage T4 replication origins.

Phage T4-like intermediates of DNA replication and recombination in the mitochondria of the higher plant Chenopodium album (L.).

We have studied intermediates of the recombination and replication of chromosomal mitochondrial (mt) DNA prepared from suspension cultured cells of Chenopodium album (L.) by electron microscopy during the whole growth cycle. We identified several types of potential recombination and replication intermediates including rosette-like structures, as well as other branched and sigma-like molecules. The absolute and relative amounts of these structures changed dramatically during the growth cycle, indicating high dynamics in the structural organization of the mt genome. The rosette-like molecules had sizes of 2-5 genome units and were found to contain putative replication forks and 'Holliday'-junctions known from recombination intermediates. The high number of rosettes during the first days of culture, and their drastic reduction in the stationary growth stage, were found to be inversely related to changes in the quantity of linear molecules of 40-200 kb. This observation suggests that linear molecules participate in the formation of giant branched rosette-like structures. Most linear molecules were previously found to have at least one single-stranded end, which may allow recombinative invasion of other double-stranded molecules. Thus, recombination events may lead to the formation of more complex molecules and initiate replication similar to phage T4. We propose the coexistence of a recombination-dependent mode of replication with a presumably recombination-independent rolling-circle mode of replication in the mitochondria of C. album.

Unique features of the mitochondrial rolling circle-plasmid mp1 from the higher plant Chenopodium album (L.).

We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mp1 (1309 bp) from the higher plant Chenopodium album(L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.

Electron microscopic investigation of mitochondrial DNA from Chenopodium album (L.).

DNA molecules from mitochondria of whole plants and a suspension culture of Chenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0. 3-183 kb represented 23-26% of all mtDNA molecules in the preparations from the suspension culture and 13-15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rolling-circle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.

Investigation of plant organellar DNAs by pulsed-field gel electrophoresis.

Mitochondrial (mt) DNAs from several higher-plant species (Arabidopsis thaliana, Beta vulgaris, Brassica hirta, Chenopodium album, Oenothera berteriana, Zea mays) were separated by pulsed-field gel electrophoresis (PFGE). Hybridization of the separated DNA with mtDNA-specific probes revealed an identical distribution of mtDNA sequences in all cases: part of the DNA formed a smear of linear molecules migrating into the gel, the rest remained in the well. Hybridization signals in the compression zone of the gels disappeared after RNase or alkaline treatment. It was shown that the linear molecules are not products of unspecific degradation by nucleases. All plastid (pt) DNA from leaves of Nicotiana tabacum remained in the well after PFGE. Separation of linear monomers and oligomers of the chloroplast chromosomes of N. tabacum was achieved by mild DNase treatment of the well-bound DNA. DNase treatment of well-bound mtDNA, however, generated a smear of linear molecules. PtDNA from cultured cells of C. album was found after PFGE to be partly well-bound, and partly separated into linear molecules with sizes of monomeric and oligomeric chromosomes. The ease with which it was possible to detect large linear molecules of plastid DNA indicates that shearing forces alone can not explain the smear of linear molecules obtained after PFGE of mtDNA. The results are discussed in relation to the structural organization of the mt genome of higher plants.

High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.).

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknown feature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22,800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.

Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells.

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti-phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).

Tyrosine phosphorylation patterns and size modification of the Helicobacter pylori CagA protein after translocation into gastric epithelial cells.

Helicobacter pylori is one of the most common bacterial pathogens that causes a variety of gastric diseases. During infection, the immuno-dominant H. pylori CagA protein is translocated and tyrosine-phosphorylated in gastric epithelial cells. We compared tyrosine phosphorylation patterns of five CagA variants by two-dimensional electrophoresis (2-DE) and immunoblotting studies. Tyrosine-phosphorylated CagA was detected as two distinct protein species in strains P12, P227, G27 and 26695 suggesting that two tyrosine residues of CagA can be phosphorylated both separately and simultaneously. Prediction programs revealed the presence of three putative tyrosine phosphorylation motifs in the sequences of CagA. Mutations in these motifs were identified suggesting that only two putative phosphorylation-relevant tyrosines are present in each CagA variant. CagA of strain J99 was found to be unique because essential codons were mutated in each of the three motifs and, consequently, revealed no tyrosine phosphorylation signals at all. These findings support the view that CagA from different H. pylori strains can be tyrosine-phosphorylated at one or two out of three predicted positions. Additionally, truncated CagA protein species of about 100-105 kDa (p100CagA) have been detected after infection with some of the H. pylori strains. The isoelectric point determined by both 2-DE and sequence analysis suggested that p100CagA represents the amino (N)-terminal part of the protein. Translocation, tyrosine phosphorylation and size modification of CagA might be involved in host signal transduction and development of gastric disease.

Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components.

Gastric infections by Helicobacter pylori are characteristically associated with an intense inflammation and infiltration of mainly polymorphonuclear lymphocytes (PMNs) and monocytes. The inflammatory response by infiltrated immune cells appears to be a primary cause of the damage to surface epithelial layers and may eventually result in gastritis, peptic ulcer, gastric cancer and/or MALT-associated gastric lymphoma. Our analysis of the interaction between H. pylori and PMNs and monocytes revealed that H. pylori inhibits its own uptake by these professional phagocytes. To some degree, this effect resembles antiphagocytosis by Yersinia enterocolitica. Increasing numbers of bacteria associated per cell are more efficient at blocking their own engulfment. In H. pylori, bacterial protein synthesis is necessary to block phagocytic uptake, as shown by the time and concentration dependence of the bacteriostatic protein synthesis inhibitor chloramphenicol. Furthermore, H. pylori appears broadly to inhibit the phagocytic function of monocytes and PMNs, as infection with H. pylori abrogates the phagocytes' ability to engulf latex beads or adherent Neisseria gonorrhoeae cells. This antiphagocytic phenotype depends on distinct virulence (vir) genes, such as virB7 and virB11, encoding core components of a putative type IV secretion apparatus. Our data indicate that H. pylori exhibits an antiphagocytic activity that may play an essential role in the immune escape of this persistent pathogen.