Virulence of Campylobacter jejuni for chicken embryos.

The pathogenicity of Campylobacter jejuni was examined in chicken embryos. In this system, mortality data and histopathological findings induced by organisms and by bacterium-free filtered broth were identical. The absence in chicken embryo tissues both of organisms and of an inflammatory infiltrate suggests a toxin etiology.     

Role of Campylobacter jejuni as a placental pathogen.

Members of the genus Campylobacter are well recognised as enteric pathogens but have rarely been implicated as human placental pathogens. A case of septic abortion due to C jejuni is reported. This occurred in a previously healthy woman with a diarrhoeal illness. The presence of chorioamnionitis raises the possibility of ascending infection rather than septicaemic spread as the pathogenesis. Members of the genus Campylobacter have special culture requirements and their incidence as placental pathogens may therefore be underestimated.     

EptC of Campylobacter jejuni Mediates Phenotypes Involved in Host Interactions and Virulence

Campylobacter jejuni is a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Like many bacterial pathogens, C. jejuni assembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in C. jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and several N-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification of C. jejuni lipid A with pEtN results in increased recognition by the human Toll-like receptor 4–myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lacking eptC show decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.     

Characterization of the Thermal Stress Response of Campylobacter jejuni

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage λ to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46°C was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.     

An Acid Extract as a Common Antigen in Campylobacter coli and Campylobacter jejuni Strains

An acid extract prepared from a single strain of Campylobacter jejuni reacted with rabbit antisera against 21 strains of C. jejuni and Campylobacter coli. The immunogen is resistant to Formalin and to exposure to 100 degrees C for 30 min. The antigenic extract may have practical applications in human serology.     

Identification of the Enteropathogens Campylobacter jejuni and Campylobacter coli Based on the cadF Virulence Gene and Its Product

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.     

Molecular detection of Campylobacter jejuni as a cause of culture-negative spondylodiscitis

Spondylodiscitis caused by Campylobacter species is a rare disease which is most often caused by Campylobacter fetus. We report a case of culture-negative spondylodiscitis and a psoas abscess due to Campylobacter jejuni in a 68-year-old woman, as revealed by 16S rRNA gene and Campylobacter-specific PCRs from biopsied tissue.     

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.     

Demonstration of a cytotoxin from Campylobacter jejuni

A 48-hour culture filtrate of Campylobacter jejuni was found to produce cytopathic effects on three human cell lines--that is, HeLa, MRC-5 and Hep-2. The cytopathic effects observed include cell rounding, loss of adherence and cell death after 24-48 h of incubation. Such morphological changes were observed with eight of the eleven strains of Campylobacter jejuni isolated from the blood/stools of patients who suffered from either acute gastroenteritis or septicaemia. The toxic factor did not retain its activity after treatment at 100 degrees C for 30 min. Trypsinisation of the filtrate totally abolished its toxic activity thus indicating that it was probably protein in nature. It is most probably an extracellular toxin as bacterial sonicates did not produce any toxic effect. This study reports the finding of toxic factor(s) in the culture filtrate of Campylobacter jejuni which is cytotoxic to human tissue culture cells.     

Characterization of a 25,000-dalton Helicobacter pylori protein, cross-reacting with a Campylobacter jejuni protein.

Extract obtained by ultrasonic disruption of Helicobacter pylori bacteria contained a protein with subunit molecular mass of 25 kD which bound antibodies in sera from patients with H. pylori-associated disease. The protein was purified by gel permeation and elution from SDS-polyacrylamide gel slices, and was used to raise an anti-25-kD protein-specific rabbit serum. Using the antiserum in experiments, the results indicated the following: The protein exists as covalently linked dimers (45 kD) of the 25-kD subunits. Variable numbers of non-covalently linked copies of the dimers make up the native protein. The protein was susceptible to digestion by papain, pronase, and trypsin. Pepsin cleaved off a fragment of approximately 2 kD. A small share of the protein was exposed at the bacterial cell surface, the greatest share being localized internally. The protein was not secreted and it was probably not an integral part of the outer membrane. It was produced in variable quantity by all of 11 H. pylori strains tested and was a major protein in some strains. A cross-reacting protein with subunit size of 25 kD was also produced by Campylobacter jejuni strains, but not by any of a variety of other bacteria. Since both H. pylori and C. jejuni infection occur with a high frequency. the cross-reacting 25-kD protein may interfere unfavourably with the diagnostic specificity of serological tests for infection caused by these bacteria.     

Characterization of the Specific Interaction between Sialoadhesin and Sialylated Campylobacter jejuni Lipooligosaccharides

In Campylobacter jejuni-induced Guillain-Barré syndrome (GBS), molecular mimicry between C. jejuni lipooligosaccharide (LOS) and host gangliosides leads to the production of cross-reactive antibodies directed against the peripheral nerves of the host. Currently, the presence of surface exposed sialylated LOS in C. jejuni is the single known bacterial pathogenesis factor associated with the development of GBS. Using a unique, well-characterized strain collection, we demonstrate that GBS-associated C. jejuni strains bind preferentially to sialoadhesin (Sn, Siglec-1, or CD169), a sialic acid receptor found on a subset of macrophages. In addition, using a whole-cell enzyme-linked immunosorbent assay (ELISA), C. jejuni strains with sialylated LOS bound exclusively to soluble Sn. Mass spectrometry revealed that binding was sialic acid-linkage specific with a preference for α(2,3)-linked sialic acid attached to the terminal galactose of the LOS chain as seen in the gangliosides GD1a, GM1b, and GM3. This molecular interaction was also related to functional consequences as a GBS-associated C. jejuni strain that bound Sn in a whole-cell ELISA adhered to surface-expressed Sn of Sn-transfected CHO cells but was unable to adhere to wild-type CHO cells. Moreover, a sialic acid-negative mutant of the same C. jejuni strain was unable to bind Sn-transfected CHO cells. This is the first report of the preferential binding of GBS-associated C. jejuni strains to the Sn immune receptor (P = 0.014). Moreover, because this binding is dependent on sialylated LOS, the main pathogenic factor in GBS progression, the present findings bring us closer to unraveling the mechanisms that lead to formation of cross-reactive antibodies in GBS disease.Campylobacter jejuni, a food-borne Gram-negative bacterium, is the major cause of bacterial gastroenteritis worldwide. In addition to enteritis, infection with C. jejuni may also lead to a neurological complication called the Guillain-Barré syndrome (GBS). GBS is an autoimmune disease affecting the peripheral nerves. Antibodies raised by the host during an infection with C. jejuni possess the capacity to cross-react with structures on human nerve tissue, resulting in neurological complications for the host (38). Further, high titers of anti-ganglioside antibodies are frequently found in the sera of GBS patients (24, 41). Gangliosides are glycosphingolipids with an extracellular sialylated oligosaccharide chain and a ceramide tail that is embedded in the outer leaflet of the plasma membrane. Although predominantly found in the nervous system, gangliosides are present on other cell surfaces as well.C. jejuni has lipooligosaccharide (LOS) structures on its outer membrane. Biochemical and structural analysis of LOS outer core oligosaccharides has identified sialylated moieties that are structurally similar to several gangliosides (6, 9, 30). During infection, the structural similarity between C. jejuni LOS and human gangliosides, also known as molecular mimicry, facilitates the induction of anti-ganglioside antibodies and the development of GBS (1, 29, 38, 40). The C. jejuni genes involved in ganglioside mimicry are located within the LOS biosynthesis locus, a gene cluster that is interchangeable between strains and is genetically highly diverse (18, 19). Therefore, several LOS classes (A through S) have been identified (31). LOS class, gene alterations, mutations, and mechanisms such as phase variation in the LOS locus contribute to structural variations in the ganglioside mimics produced (19). The presence of LOS biosynthesis locus-encoded genes responsible for synthesis, modification, and transfer of sialic acid, found in LOS classes A, B, and C, is crucial in the induction of anti-ganglioside antibodies and hence GBS (20, 36). Sialylated LOS is also involved in other aspects of C. jejuni pathogenesis. C. jejuni strains expressing sialylated LOS invade human epithelial intestinal cells significantly more frequently than strains expressing nonsialylated LOS (28). However, the receptor for C. jejuni attachment to human epithelial intestinal cells is unknown.Certain C. jejuni strains are known to bind to Siglec-7, a member of the sialic acid binding immunoglobulin-like lectin (Siglec) family (8). Siglecs are present on the cell surface of a range of immune-associated cells and are involved in cell to cell interactions and signaling. A subset of the Siglec family, the CD33-related Siglecs, can serve as regulators of the immune system through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tail (7, 11). In addition, several recently described human Siglecs, Siglecs-14, -15, and -16 can interact with the immunoreceptor tyrosine-based activation motif (ITAM) adaptor, DAP12 and therefore potentially mediate the activation of intracellular signaling (5, 10).Sialoadhesin (Sn, Siglec-1, or CD169) is a macrophage-restricted Siglec that has been associated with inflammatory and autoimmune diseases. For example, Sn levels are elevated on activated macrophages within the inflamed organs of several inflammatory disorders, including rheumatoid arthritis, experimental autoimmune encephalomyelitis (EAE), and experimental autoimmune uveoretinitis (EAU) (22, 32, 39). This elevated expression may have functional consequences since Sn-deficient mice show a reduced severity of EAE and EAU (25, 39). With a poorly conserved cytoplasmic tail and the absence of tyrosine-based signaling motifs, Sn seems to be more involved in cell-to-cell communication and ligand binding than intracellular immunoregulation. It has been shown that macrophages expressing Sn can bind and internalize sialylated Neisseria meningitidis in an Sn- and sialic acid-dependent manner (26). Further, HIV-1 can interact with Sn, probably via a sialic acid residue on gp120, with binding resulting in enhanced infectivity and facilitates transinfections in alpha interferon (IFN-α)-stimulated CD14⁺ monocytes. Furthermore, Sn gene expression is elevated in CD14⁺ monocytes from patients infected with HIV-1 (34).Each Siglec has a unique specificity for certain sialylated glycans, with Sn preferring sialic acid conjugates with an α(2,3)galactose (gal) linkage (12). This α(2,3)Gal linkage is often found on the LOS of GBS-associated C. jejuni strains.Because of the connection of C. jejuni infection with autoimmune disease such as GBS and its clinical variant Miller-Fisher syndrome (MFS), we investigated whether α(2,3)-linked sialic acid residues on the surface of C. jejuni strains could interact with Sn and whether this interaction was characteristic for GBS-associated strains.     

Characterization of two Campylobacter jejuni strains for use in volunteer experimental-infection studies

The development of vaccines against Campylobacter jejuni would be facilitated by the ability to perform phase II challenge studies. However, molecular mimicry of the lipooligosaccharide (LOS) of most C. jejuni strains with human gangliosides presents safety concerns about the development of Guillain-Barré syndrome. Clinical isolates of C. jejuni that appeared to lack genes for the synthesis of ganglioside mimics were identified by DNA probe analyses. Two clinical isolates from Southeast Asia (strains BH-01-0142 and CG8421) were determined to express the LOS type containing N-acetyl quinovosamine. No ganglioside structures were observed to be present in the LOSs of these strains, and pyrosequence analyses of the genomes of both strains confirmed the absence of genes involved in ganglioside mimicry. The capsule polysaccharide (CPS) of BH-01-0142 was determined to be composed of galactose (Gal), 6-deoxy-ido-heptose, and, in smaller amounts, D-glycero-D-ido-heptose, and the CPS of CG8421 was observed to contain Gal, 6-deoxy-altro-heptose, N-acetyl-glucosamine, and minor amounts of 6-deoxy-3-O-Me-altro-heptose. Both CPSs were shown to carry O-methyl-phosphoramidate. The two genomes contained strain-specific zones, some of which could be traced to a plasmid origin, and both contained a large chromosomal insertion related to the CJEI3 element of C. jejuni RM1221. The genomes of both strains shared a high degree of similarity to each other and, with the exception of the capsule locus of CG8421, to the type strain of the HS3 serotype, TGH9011.     

The gnotobiotic piglet as a model for the pathogenesis of Campylobacter jejuni infection

The pathogenesis of enteric changes was studied in gnotobiotic piglets which, after hysterectomy had been infected orally with Campylobacter jejuni on the first day of their life. The involvement of the entire large intestine became clinically manifest by scouring on days post infection (DPI) 4 to DPI 5, and pathomorphologically, by simultaneous inflammation and severe edema of the intestinal wall. Histology and SEM revealed inflammatory edema with abundant neutrophils, microulcerations, focal propagation and activation of goblet cells, and a presence of mucin-positive material within the intestinal lumen. TEM examination revealed disconnected interdigitating folds and wide dilated intercellular spaces between enterocytes. The endothelial cells of small blood vessels in the lamina propria showed hypertrophy with increase in the thickness of their basal lamina. Ultrastructural lesions of the large intestinal microcirculation also support the hypothesis that disturbances in the vascular system are responsible for edema in the cecum and colon. Gnotobiotic piglets may be used as a suitable animal model to study colitis induced by C. jejuni.     

Hepatotoxic activity of Campylobacter jejuni

Hepatotoxic factor(s) were isolated from whole-cell lysates of Campylobacter jejuni GIFU 8734 and purified by chromatography. A single intravenous injection of 10 micrograms of this factor reproducibly produced hepatitis in mice, as determined by histology and liver function tests. The hepatic lesions were very similar to those evoked by C. jejuni infection. Tissue-culture studies with mouse hepatocytes demonstrated that low concentrations of the factor caused release of hepatic enzymes into the medium without appreciable cytolysis. High concentrations of the factor induced cytolysis. These effects were neutralised by antiserum to the factor, but not by antisera to the lipopolysaccharide of C. jejuni or to the heat-labile enterotoxin of Escherichia coli. Among 20 clinical isolates of C. jejuni, only four evoked hepatitis in mice and produced the hepatotoxic factor.     

Fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni

The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.     

Development of a bacteriophage typing system for Campylobacter jejuni and Campylobacter coli.

A bacteriophage typing system for Campylobacter jejuni and Campylobacter coli was developed with phages isolated from poultry feces. Data for phage selection were generated from a set of isolates of C. jejuni and C. coli from humans in Illinois. Selection of 14 phages from the 47 phages available was assisted by determination of the Sneath-Jaccard similarity coefficients and subsequent unweighted pair-group arithmetic averaging cluster analysis. The typing set was reproducible and stable in the 255 isolates from Illinois. Of these isolates, 94.5% were typable, with 46% represented by the four most common phage patterns. In a set of 51 isolates from humans outside of Illinois, 88.1% of the C. jejuni isolates were typable. Phage typing for C. jejuni and C. coli has excellent epidemiologic potential and should serve as a useful adjunct or alternative to serotyping systems in current use.     

Unique features of a highly pathogenic Campylobacter jejuni strain

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain.