Cell-surface alpha-glucan in Campylobacter jejuni 81-176

Campylobacter jejuni infection is a main source of severe gastroenteritis-related illnesses in humans and there is also evidence that it may be linked to neurological disorders. C. jejuni 81-176 is a virulent strain that has become the global model in the study of mechanisms and pathogenesis of C. jejuni infection. For this reason, we were engaged in studying the fine structures of cell-surface carbohydrate antigens of C. jejuni 81-176, namely, the capsule polysaccharide (CPS) and lipooligosaccharide (LOS). Serologically, C. jejuni 81-176 has been classified as belonging to serogroups HS23 and HS36, and indeed previous studies have shown that the LOS and CPS structures possess components similar to those expressed by serostrains HS23 and HS36. Here, we describe that in addition to the LOS and CPS, this strain also produced an independent cell-surface (1-->4)-alpha-glucan capsule.     

Crystal structure of the Campylobacter jejuni CmeC outer membrane channel

As one of the world's most prevalent enteric pathogens, Campylobacter jejuni is a major causative agent of human enterocolitis and is responsible for more than 400 million cases of diarrhea each year. The impact of this pathogen on children is of particular significance. Campylobacter has developed resistance to many antimicrobial agents via multidrug efflux machinery. The CmeABC tripartite multidrug efflux pump, belonging to the resistance‐nodulation‐cell division (RND) superfamily, plays a major role in drug resistant phenotypes of C. jejuni. This efflux complex spans the entire cell envelop of C. jejuni and mediates resistance to various antibiotics and toxic compounds. We here report the crystal structure of C. jejuni CmeC, the outer membrane component of the CmeABC tripartite multidrug efflux system. The structure reveals a possible mechanism for substrate export.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery

Campylobacter jejuni is one of the most intriguing human foodborne bacterial pathogen. Its survival throughout the food processing chain and its pathogenesis mechanisms in humans remain enigmatic. Living in the animal guts and particularly in avian intestine as a commensal bacterium, this microorganism is frequently isolated from meat products. Ultra high pressure (HP) is a promising alternative to thermal technology for microbial safety of foodstuffs with less organoleptic and nutritional alterations. Its application could be extended to meat products potentially contaminated by C. jejuni. To evaluate the response of Campylobacter to this technological stress and subsequent recovery at a molecular level, a dynamic 2-DE-based proteomic approach has been implemented. After cultivation, C. jejuni cells were conditioned in a high-pressure chamber and transferred to fresh medium for recovery. The protein abundance dynamics at the proteome scale were analyzed by 2-DE during the cellular process of cell injury and recovery. Monitoring protein abundance through time unraveled the basic metabolisms involved in this cellular process. The significance of the proteome evolution modulated by HP and subsequent recovery is discussed in the context of a specific cellular response to stress and recovery of C. jejuni with 69 spots showing significant changes through time.     

Structural context for protein N-glycosylation in bacteria: The structure of PEB3, an adhesin from Campylobacter jejuni

Campylobacter jejuni is unusual among bacteria in possessing a eukaryotic-like system for N-linked protein glycosylation at Asn residues in sequons of the type Asp/Glu-Xaa-Asn-Xaa-Ser/Thr. However, little is known about the structural context of the glycosylated sequons, limiting the design of novel recombinant glycoproteins. To obtain more information on sequon structure, we have determined the crystal structure of the PEB3 (Cj0289c) dimer. PEB3 has the class II periplasmic-binding protein fold, with each monomer having two domains with a ligand-binding site containing citrate located between them, and overall resembles molybdate- and sulfate-binding proteins. The sequon around Asn90 is located within a surface-exposed loop joining two structural elements. The three key residues are well exposed on the surface; hence, they may be accessible to the PglB oligosaccharyltransferase in the folded state.     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Characterization of the biochemical properties of Campylobacter jejuni RNase III

Campylobacter jejuni is a foodborne bacterial pathogen, which is now considered as a leading cause of human bacterial gastroenteritis. The information regarding ribonucleases in C. jejuni is very scarce but there are hints that they can be instrumental in virulence mechanisms. Namely, PNPase (polynucleotide phosphorylase) was shown to allow survival of C. jejuni in refrigerated conditions, to facilitate bacterial swimming, cell adhesion, colonization and invasion. In several microorganisms PNPase synthesis is auto-controlled in an RNase III (ribonuclease III)-dependent mechanism. Thereby, we have cloned, overexpressed, purified and characterized Cj-RNase III (C. jejuni RNase III). We have demonstrated that Cj-RNase III is able to complement an Escherichia coli rnc-deficient strain in 30S rRNA processing and PNPase regulation. Cj-RNase III was shown to be active in an unexpectedly large range of conditions, and Mn²⁺ seems to be its preferred co-factor, contrarily to what was described for other RNase III orthologues. The results lead us to speculate that Cj-RNase III may have an important role under a Mn²⁺-rich environment. Mutational analysis strengthened the function of some residues in the catalytic mechanism of action of RNase III, which was shown to be conserved.     

Alternative bacteriophage life cycles: the carrier state of Campylobacter jejuni

Members of the genus Campylobacter are frequently responsible for human enteric disease, often through consumption of contaminated poultry products. Bacteriophages are viruses that have the potential to control pathogenic bacteria, but understanding their complex life cycles is key to their successful exploitation. Treatment of Campylobacter jejuni biofilms with bacteriophages led to the discovery that phages had established a relationship with their hosts typical of the carrier state life cycle (CSLC), where bacteria and bacteriophages remain associated in equilibrium. Significant phenotypic changes include improved aerotolerance under nutrient-limited conditions that would confer an advantage to survive in extra-intestinal environments, but a lack in motility eliminated their ability to colonize chickens. Under these circumstances, phages can remain associated with a compatible host and continue to produce free virions to prospect for new hosts. Moreover, we demonstrate that CSLC host bacteria can act as expendable vehicles for the delivery of bacteriophages to new host bacteria within pre-colonized chickens. The CSLC represents an important phase in the ecology of Campylobacter bacteriophage.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

RpoD promoters in Campylobacter jejuni exhibit a strong periodic signal instead of a -35 box

We have used a hidden Markov model (HMM) to identify the consensus sequence of the RpoD promoters in the genome of Campylobacter jejuni. The identified promoter consensus sequence is unusual compared to other bacteria, in that the region upstream of the TATA-box does not contain a conserved -35 region, but shows a very strong periodic variation in the AT-content and semi-conserved T-stretches, with a period of 10-11 nucleotides. The TATA-box is in some, but not all cases, preceded by a TGx, similar to an extended -10 promoter.We predicted a total of 764 presumed RpoD promoters in the C.jejuni genome, of which 654 were located upstream of annotated genes. A similar promoter was identified in Helicobacter pylori, a close phylogenetic relative of Campylobacter, but not in Escherichia coli, Vibrio cholerae, or six other Proteobacterial genomes, or in Staphylococcus aureus. We used upstream regions of high confidence genes as training data (n=529, for the C.jejuni genome). We found it necessary to limit the training set to genes that are preceded by an intergenic region of >100bp or by a gene oriented in the opposite direction to be able to identify a conserved sequence motif, and ended up with a training set of 175 genes. This leads to the conclusion that the remaining genes (354) are more rarely preceded by a (RpoD) promoter, and consequently that operon structure may be more widespread in C.jejuni than has been assumed by others.Structural predictions of the regions upstream of the TATA-box indicates a region of highly curved DNA, and we assume that this facilitates the wrapping of the DNA around the RNA polymerase holoenzyme, and offsets the absence of a conserved -35 binding motif.     

Molecular cloning and transposition of a kanamycin resistance determinant from Campylobacter jejuni between replicons in Escherichia coli

This paper reports a restriction map of a fragment of DNA encoding kanamycin resistance cloned from plasmid DNA of Campylobacter jejuni ABA94 in the recombinant plasmid pRS9421-1. In transposition experiments, kanamycin-resistant R751::km9421 transconjugants appeared at frequencies of 10^-7 per donor cell. These transconjugants harboured a plasmid 4 kb larger than the parental 49 kb plasmid R751. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that the kanamycin resistant determinant had transposed from recombinant plasmid pRS9421-1 to plasmid R751.The authors are with the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering

Conjugate vaccines belong to the most efficient preventive measures against life-threatening bacterial infections. Functional expression of N-oligosaccharyltransferase (N-OST) PglB of Campylobacter jejuni in Escherichia coli enables a simplified production of glycoconjugate vaccines in prokaryotic cells. Polysaccharide antigens of pathogenic bacteria can be covalently coupled to immunogenic acceptor proteins bearing engineered glycosylation sites. Transfer efficiency of PglB_Cj is low for certain heterologous polysaccharide substrates. In this study, we increased glycosylation rates for Salmonella enterica sv. Typhimurium LT2 O antigen (which lacks N-acetyl sugars) and Staphylococcus aureus CP5 polysaccharides by structure-guided engineering of PglB. A three-dimensional homology model of membrane-associated PglB_Cj, docked to the natural C. jejuni N-glycan attached to the acceptor peptide, was used to identify potential sugar-interacting residues as targets for mutagenesis. Saturation mutagenesis of an active site residue yielded the enhancing mutation N311V, which facilitated fivefold to 11-fold increased in vivo glycosylation rates as determined by glycoprotein-specific ELISA. Further rounds of in vitro evolution led to a triple mutant S80R-Q287P-N311V enabling a yield improvement of S. enterica LT2 glycoconjugates by a factor of 16. Our results demonstrate that bacterial N-OST can be tailored to specific polysaccharide substrates by structure-guided protein engineering.