Helicobacter pylori lipopolysaccharide is synthesized via a novel pathway with an evolutionary connection to protein N-glycosylation.

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.

Campylobacter jejuni drives MyD88-independent interleukin-6 secretion via Toll-like receptor 2.

Gastrointestinal disease caused by Campylobacter jejuni is characterized by localized inflammation and the destruction of the epithelial cell barrier that forms host innate protection against pathogens. This can lead to an imbalance in fluid transport across the gastrointestinal tract, resulting in severe diarrhea. The mechanisms of host cell receptor recognition of C. jejuni and downstream immune signaling pathways leading to this inflammatory disease, however, remain unclear. The aim of this study was to analyze the mechanisms involved in C. jejuni induction of the acute-phase inflammatory response regulator interleukin-6 (IL-6). Polarized intestinal epithelial Caco-2 monolayers responded to infections with Salmonella enterica serovar Typhimurium and eight isolates of C. jejuni by an increase in levels of expression and secretion of IL-6. No such IL-6 response, however, was produced upon infection with the human commensal organism Lactobacillus rhamnosus GG. The IL-6 signaling pathway was further characterized using short interfering RNA complexes to block gene expression. The inhibition of myeloid differentiation primary response protein 88 (MyD88) expression in this manner did not affect C. jejuni-induced IL-6 secretion, suggesting a MyD88-independent route to IL-6 signal transduction in C. jejuni-infected human epithelial cells. However, a significant reduction in levels of IL-6 was evident in the absence of Toll-like receptor 2 (TLR-2) expression, implying a requirement for TLR-2 in C. jejuni recognition. Caco-2 cells were also treated with heat-inactivated and purified membrane components of C. jejuni to isolate the factor responsible for triggering IL-6 signaling. The results demonstrate that C. jejuni surface polysaccharides induce IL-6 secretion from intestinal epithelial cells via TLR-2 in a MyD88-independent manner.

Chimeras of bacterial translation factors Tet(O) and EF-G.

Ribosomal protection proteins (RPPs) confer bacterial resistance to tetracycline by releasing this antibiotic from ribosomes stalled in protein synthesis. RPPs share structural similarity to elongation factor G (EF-G), which promotes ribosomal translocation during normal protein synthesis. We constructed and functionally characterized chimeric proteins of Campylobacter jejuni Tet(O), the best characterized RPP, and Escherichia coli EF-G. A distinctly conserved loop sequence at the tip of domain 4 is required for both factor-specific functions. Domains 3-5: (i) are necessary, but not sufficient, for functional specificity; and (ii) modulate GTP hydrolysis by EF-G, while minimally affecting Tet(O), under substrate turnover conditions.

Species identification of erythromycin-resistant Campylobacter isolates and optimization of a duplex PCR for rapid detection.

This study describes the approach used to verify the species identity of 23 erythromycin-resistant Campylobacter isolates whose identity was initially determined based mainly on the results of the rapid hippurate hydrolysis test or the results of the API-Campy identification system. Species identification of the isolates investigated was confirmed by repeating hippurate hydrolysis using a modification of the rapid hydrolysis test, in addition to performing three genetic-based assays. The original identification was verified in 69.6% of the isolates. The remaining isolates showed discrepancies in identity as determined by results of the identification assays performed. A duplex PCR assay, targeting the hipO and aspA genes, indicated the existence of mixed cultures of C. jejuni and C. coli in the frozen stocks of two of these isolates.

A novel Helicobacter pylori cell-surface polysaccharide.

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.

Molecular biology methods for the characterization of Helicobacter pylori infections and their diagnosis.

Helicobacter pylori infects approximately half of the human population; however, the outcome of infection is affected by many factors, including strain and host genotype characteristics and bacterial density within the stomach. Many molecular methods have been developed to provide information with respect to these characteristics. Methods that provide results within 24 h of endoscopy may be used to develop individualized treatment that is more effective, results in fewer side effects, cuts costs,decreases the number of treatment failures and results in the development of fewer antibiotic-resistant H. pylori strains.

Characterization of plasmid-mediated aphA-3 kanamycin resistance in Campylobacter jejuni.

A total of 254 isolates of Campylobacter jejuni and three isolates of Campylobacter coli, isolated from Sweden, Canada, and Egypt, were screened for kanamycin resistance. Eight strains of C. jejuni contained large plasmids that carried the aphA-3 kanamycin-resistance marker. In six plasmids, the aphA-3 gene was located downstream of an apparent insertion sequence, designated IS607*, which showed a considerable similarity to IS607, characterized on the chromosome of some Helicobacter pylori strains. In contrast, the other plasmids carried the aphA-3 gene as a part of a resistance cluster. This included three resistance markers encoding 6'-adenylyltransferase (aadE), streptothricin acetyltransferase (sat), and 3'-aminoglycoside phosphotransferase type III (aphA-3). The genetic organization of this resistance cluster suggests that it has been acquired by C. jejuni from a Gram-positive organism. The IS607* element was also observed in kanamycin-susceptible strains of C. jejuni on plasmids mediating tetracycline resistance. The kanamycin-resistance phenotype transferred along with tetracycline resistance by conjugation from four representative C. jejuni strains to a recipient strain of C. jejuni. The kanamycin-resistance determinant (aphA-3) was stably transferred from one of the four C. jejuni strains to a recipient strain of Escherichia coli. However, the C. jejuni plasmid, which also carries the tetO gene, was not maintained in E. coli. Pulsed-field gel electrophoresis revealed the integration of approximately 50 kb of the plasmid into the chromosome of the E. coli recipient.

Entry exclusion in the IncHI1 plasmid R27 is mediated by EexA and EexB.

Conjugative plasmids have evolved entry exclusion mechanisms to inhibit redundant DNA transfer from donor cells into recipients harboring isogenic or closely related plasmids. This exclusion phenomenon has been documented in the incompatibility H group (IncH) plasmid R27. A cosmid library representing the majority of the large (180kb) R27 plasmid was transformed into recipient cells and a conjugation assay identified that an operon located in the conjugative transfer region 2 (Tra2) of R27, the Z operon, mediated entry exclusion in the IncH plasmid. Reverse-transcriptase analysis revealed that the Z operon is comprised of four genes, 015, eexB, 017, and eexA. Sub-cloning of the individual genes located within the Z operon and subsequent screening for the entry exclusion phenotype determined that two genes, eexA and eexB, independently inhibit the entry of IncH-related plasmids. Bacterial fractionation studies predominantly localized the EexA protein to the cytoplasmic membrane, and the EexB protein to the outer membrane. Recipient cells expressing EexA and EexB were unable to exclude the entry of R27 plasmids harboring mutations within the IncH entry exclusion genes eexA and eexB. The IncH entry exclusion proteins EexA and EexB likely prevent redundant plasmid transfer by interaction with one another.

Contribution of the CmeABC efflux pump to macrolide and tetracycline resistance in Campylobacter jejuni.

We investigated the involvement of the CmeABC efflux pump in acquired resistance of Campylobacter jejuni to macrolides and tetracycline. Inactivation of the cmeB gene had no effect on macrolide resistance when all copies of the target gene carried an A2074C mutation. In contrast, the CmeABC pump significantly contributed to macrolide resistance when two or three copies of the 23S rRNA had an A2075G transition. Inactivation of the cmeB gene led to restoration of tetracycline susceptibility in the isolates examined. Complete susceptibility to tetracycline or macrolides, however, was not restored when phenylalanine-arginine beta-naphthylamide was used. These data confirm contribution of the CmeABC efflux pump to acquired resistance of Campylobacter jejuni to tetracycline and macrolides.

Interaction between the co-inherited TraG coupling protein and the TraJ membrane-associated protein of the H-plasmid conjugative DNA transfer system resembles chromosomal DNA translocases.

Bacterial conjugation is a DNA transfer event that requires three plasmid-encoded multi-protein complexes: the membrane-spanning mating pair formation (Mpf) complex, the cytoplasmic nucleoprotein relaxosome complex, and a homo-multimeric coupling protein that links the Mpf and relaxosome at the cytoplasmic membrane. Bacterial two-hybrid (BTH) technology and immunoprecipitation were used to demonstrate an interaction between the IncH plasmid-encoded transfer protein TraJ and the coupling protein TraG. TraJ is essential for conjugative transfer but is not required for the formation of the conjugative pilus, and is therefore not regarded as an Mpf component. Fractionation studies indicated that TraJ shared a similar cellular domain to that of TraG at the cellular membrane. Protein blast analyses have previously identified TraJ homologues encoded in a multitude of plasmid and chromosomal genomes that were also found to encode an adjacent TraG homologue, thus indicating co-inheritance. BTH analysis of these TraJ and cognate TraG homologues demonstrated conservation of the TraJ-TraG interaction. Additional occurrences of the traJ-traG module were also detected in genomic sequence data throughout the Proteobacteria, and phylogenetic comparison of these IncH-like TraG proteins with the coupling proteins encoded by other conjugative transfer systems (including IncP, IncW and IncF) that lack TraJ homologues indicated that the H-like coupling proteins were distinct. Accordingly, the IncP, IncW and IncF coupling proteins were unable to interact with TraJ, but were able to interact with IncH plasmid-encoded TrhB, an Mpf component known to complex with its cognate coupling protein TraG. The divergence of the IncH-type coupling proteins may partly be due to the requirement of TraJ interaction, and notably, TraG and TraJ cumulatively represent the domain architecture of the known translocase family FtsK/SpoIIIE. It is proposed that TraJ is a functional part of the IncH-type coupling protein complex required for translocation of DNA through the cytoplasmic membrane.

An improved procedure for expression and purification of ribosomal protection protein Tet(O) for high-resolution structural studies.

Tetracycline (Tc) is a broad spectrum antibiotic that binds to the A site of the bacterial ribosome inhibiting delivery of aminoacyl-tRNA to the A site for productive protein biosynthesis. Tet(O) is in a class of the ribosomal protection proteins (RPPs) found in many pathogenic bacteria, that dislodges Tc from the A site of 70S ribosome to restore polypeptide elongation and confer Tc resistance to the bacteria. Considerable difficulty has been encountered in overexpressing and purifying Tet(O) from various Escherichia coli strains using lambdaPI, tac or T7 promoters. Here we report molecular cloning, overexpression of His-tagged Tet(O) in E. coli, an improved purification procedure and initial biochemical and biophysical characterization of His-tagged Tet(O).

Macrolide resistance in Campylobacter jejuni and Campylobacter coli.

Infection with Campylobacter jejuni is now considered to be the most common cause of acute bacterial gastroenteritis in humans worldwide. It occurs more frequently than infections caused by Salmonella species, Shigella species, or Escherichia coli O157:H7. Although C. jejuni is also recognized for its association with serious post-infection neurological complications, most patients with C. jejuni infections have a self-limited illness. Nevertheless, a substantial proportion of these infections are treated with antibiotics. These include severe and prolonged cases of enteritis, infections in immune-suppressed patients, septicaemia and other extra-intestinal infections. Under these circumstances, erythromycin is often recommended as the drug of first choice. However, erythromycin-resistant Campylobacter have emerged during therapy with macrolides. Moreover, the widespread use of macrolides, including erythromycin, in veterinary medicine has accelerated this resistance trend. Several countries including Canada, Japan and Finland have reported C. jejuni isolates with low and stable rates of macrolide resistance. In contrast, the increasing level of macrolide resistance in C. jejuni is becoming a major public health concern in other parts of the world such as the United States, Europe and Taiwan. Macrolide resistance in Campylobacter is mainly associated with point mutation(s) occurring in the peptidyl-encoding region in domain V of the 23S rRNA gene, the target of macrolides. Several rapid and practical techniques have recently been developed for the identification of macrolide-resistant isolates of C. jejuni. The aim of this mini-review is to give an overview of the worldwide distribution of macrolide resistance in C. jejuni and Campylobacter coli as well as its possible association with the massive use of these agents in food animals. Mechanisms implicated in macrolide resistance in C. jejuni and also techniques that have been developed for the efficient detection of macrolide-associated mutation(s) will be discussed in detail.

Fucosylation in prokaryotes and eukaryotes.

Fucosylated carbohydrate structures are involved in a variety of biological and pathological processes in eukaryotic organisms including tissue development, angiogenesis, fertilization, cell adhesion, inflammation, and tumor metastasis. In contrast, fucosylation appears less common in prokaryotic organisms and has been suggested to be involved in molecular mimicry, adhesion, colonization, and modulating the host immune response. Fucosyltransferases (FucTs), present in both eukaryotic and prokaryotic organisms, are the enzymes responsible for the catalysis of fucose transfer from donor guanosine-diphosphate fucose to various acceptor molecules including oligosaccharides, glycoproteins, and glycolipids. To date, several subfamilies of mammalian FucTs have been well characterized; these enzymes are therefore delineated and used as models. Non-mammalian FucTs that possess different domain construction or display distinctive acceptor substrate specificity are highlighted. It is noteworthy that the glycoconjugates from plants and schistosomes contain some unusual fucose linkages, suggesting the presence of novel FucT subfamilies as yet to be characterized. Despite the very low sequence homology, striking functional similarity is exhibited between mammalian and Helicobacter pylori alpha1,3/4 FucTs, implying that these enzymes likely share a conserved mechanistic and structural basis for fucose transfer; such conserved functional features might also exist when comparing other FucT subfamilies from different origins. Fucosyltranferases are promising tools used in synthesis of fucosylated oligosaccharides and glycoconjugates, which show great potential in the treatment of infectious and inflammatory diseases and tumor metastasis.

Antimicrobial susceptibilities of Campylobacter jejuni isolates from poultry from Alberta, Canada.

One hundred four isolates of Campylobacter jejuni from poultry in Alberta, Canada, collected during 2001 were tested for resistance to 10 antimicrobial agents using agar dilution. This study provides a baseline of resistance profiles and the mechanisms of resistance observed in C. jejuni in poultry from Alberta, Canada.

Campylobacter curvus-associated hepatic abscesses: a case report.

Campylobacter curvus was isolated from blood cultures of a patient with liver abscesses. Bacterial identification involved Gram staining, biochemical analysis, gas-liquid chromatography, and 16S rRNA sequencing. The difficulty in isolation, identification, and growth of the species confirms previous work that these organisms may be overlooked by conventional detection methods.

Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases.

The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.

16S rRNA mutations that confer tetracycline resistance in Helicobacter pylori decrease drug binding in Escherichia coli ribosomes.

Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

Transcriptional organization of the temperature-sensitive transfer system from the IncHI1 plasmid R27.

One of the characteristic features of IncHI1 plasmids is a thermosensitive process of conjugation, which is optimal between 22 degrees C and 30 degrees C but inhibited at 37 degrees C. R27, the prototypical IncHI1 plasmid, contains transfer genes clustered in two regions of the plasmid, Tra1 and Tra2. In the present study, transcriptional analyses of the tra genes were undertaken at both 30 degrees C and 37 degrees C. Screening of 38 tra genes showed that tra genes are transcriptionally linked in six operons, three in each Tra region. RT-PCR analysis showed that gene expression was reduced at 37 degrees C relative to that observed at 30 degrees C. The transcription start sites of the six transcripts were identified, promoters and upstream regions were cloned, and transcription was tested at both temperatures. In cells grown at 37 degrees C, in the presence of R27, the promoters were inhibited, except for promoters of the H operon and AN operon. Conditions that influenced DNA topology, such as osmolarity, anaerobiosis, quorum sensing and acidity, showed no significant influence on transfer frequency. These results should facilitate future understanding of the basis of temperature-sensitive transfer in this large conjugative plasmid.

The Helicobacter pylori MutS protein confers protection from oxidative DNA damage.

The human gastric pathogenic bacterium Helicobacter pylori lacks a MutSLH-like DNA mismatch repair system. Here, we have investigated the functional roles of a mutS homologue found in H. pylori, and show that it plays an important physiological role in repairing oxidative DNA damage. H. pylori mutS mutants are more sensitive than wild-type cells to oxidative stress induced by agents such as H2O2, paraquat or oxygen. Exposure of mutS cells to oxidative stress results in a significant ( approximately 10-fold) elevation of mutagenesis. Strikingly, most mutations in mutS cells under oxidative stress condition are G:C to T:A transversions, a signature of 8-oxoguanine (8-oxoG). Purified H. pylori MutS protein binds with a high specific affinity to double-stranded DNA (dsDNA) containing 8-oxoG as well as to DNA Holliday junction structures, but only weakly to dsDNA containing a G:A mismatch. Under oxidative stress conditions, mutS cells accumulate higher levels (approximately threefold) of 8-oxoG DNA lesions than wild-type cells. Finally, we observe that mutS mutant cells have reduced colonization capacity in comparison to wild-type cells in a mouse infection model.