Complete Genome Sequence of Campylobacter jejuni Strain 12567, a Livestock-Associated Clade Representative.

We report here the complete genome sequence of Campylobacter jejuni strain 12567, a member of a C. jejuni livestock-associated clade that expresses glycoconjugates associated with improved gastrointestinal tract persistence.

Complete Genome Sequences of Three Campylobacter jejuni Phage-Propagating Strains.

Bacteriophage therapy can potentially reduce Campylobacter jejuni numbers in livestock, but it requires a detailed understanding of phage-host interactions. C. jejuni strains readily infected by certain phages are designated as phage-propagating strains. Here, we report the complete genome sequences of three such strains, NCTC 12660, NCTC 12661, and NCTC 12664.

Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens.

Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni "supercolonizer mutant" with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.IMPORTANCECampylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

Evidence for a system of general protein glycosylation in Campylobacter jejuni.

A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5alpha containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.

Interactions between Campylobacter jejuni and lipids.

We previously showed that motility plays several key roles in Campylobacter jejuni pathogenesis, including increasing the efficiency of C. jejuni attachment to host epithelial cells. To further characterize C. jejuni attachment, we first examined the role of carbohydrates. Experiments with Chinese hamster ovary (CHO) cell mutants with defined defects in complex carbohydrate biosynthesis revealed that oligosaccharide sequences probably play a subordinate role in C. jejuni attachment to eukaryotic cells. Simple sugars such as mannose, fucose, glucose, N-acetylglucosamine, maltose, and galactose also did not significantly alter the binding of C. jejuni to CHO cells. Thin-layer chromatography overlay analysis with lipids extracted from CHO cells suggested that C. jejuni binds to lipids. Lipid binding was further investigated using a receptor-based enzyme-linked immunosorbent assay. Hydrophobic interactions were determined to play a minor role in binding, since tetramethylurea, a strong inhibitor of hydrophobic interactions, did not significantly decrease binding between C. jejuni and lipids. The interaction was dissected further by comparing the binding of C. jejuni to lipids and their derivatives. The results showed that binding was greatest to the entire lipid structure and decreased in affinity when portions of the lipid were removed. Thin-layer chromatography overlay analysis showed that lipids with unsaturated fatty acids were bound with the highest affinity. Our results suggest that C. jejuni may interact with lipids in host cell membranes. However, lipids only partially inhibited C. jejuni binding to CHO cells, suggesting that multiple interactions occur between the bacteria and host cells.

Campylobacter jejuni motility and invasion of Caco-2 cells.

We investigated the influence of motility on Campylobacter jejuni binding and invasion of Caco-2 cells. C. jejuni was motile in soft agar at basic (pH 8.5) and neutral pH values representative of the intestinal environment. However, C. jejuni was immobilized at pH 5.0. The inability of C. jejuni to swarm on soft agar at pH 5.0 was not related to flagellar depolymerization or loss of viability. In tissue culture medium, C. jejuni displayed typical periods of straight swimming punctuated by tumbling behavior. This behavior was altered when the viscosity of the medium was adjusted to mimic the viscosity of intestinal mucus. C. jejuni showed longer periods of straight swimming with significantly increased velocity followed by pauses instead of tumbles. The binding and invasion of C. jejuni in Caco-2 cells also increased significantly in high-viscosity growth medium. We speculate that the swimming behavior of C. jejuni in a viscous environment may be an important factor in the interaction of these organisms with host epithelial cells. The pH, which affects C. jejuni motility, may also influence the tropism of these organisms.

Peptide amidation in an invertebrate: purification, characterization, and inhibition of peptidylglycine alpha-hydroxylating monooxygenase from the heads of honeybees (Apis mellifera).

Peptidylglycine alpha-hydroxylating monooxygenase (PHM), an enzyme involved in formation of neuropeptides with a C-terminal amide functionality in mammals and amphibians, was isolated from the head of an invertebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% overall yield. The bee PHM has a molecular weight of 71,000, is membrane associated but can be solubilized with a detergent (n-octyl-beta-D-glucopyranoside), and cross-reacts with rabbit antibodies generated toward bacterially expressed rat PHM. In the presence of copper, oxygen, and ascorbic acid, the enzyme hydroxylates model tripeptides such as dansyl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with retention of configuration. Using this tripeptide as substrate, the Km is 1.7 microM and the Vmax is 2.3 nmol.micrograms-1.h-1. Treatment of the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue and mechanism-based inactivator of PHM from pig pituitary, results in irreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe-L-vinylglycine, is only a competitive inhibitor (IC50 = 320 microM).

Malignant teratoid medulloepithelioma in a horse.

Enlargement of the left eye, corneal opacification, and blindness were clinical signs of a teratoid medulloepithelioma in a 5-year-old Standardbred mare. Diagnosis was made on histologic examination of the enucleated eye. Medulloepitheliomas are congenital intraocular tumors arising from primitive ciliary body epithelium. Their development is rare in domestic animals.