Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.     

Toxic megacolon as a complication of Campylobacter jejuni enterocolitis

We report the case of a previously healthy 53-year-old white male who developed an extraordinary complication of acute Campylobacter jejuni colitis. Toxic megacolon occurred while the patient was treated with a fluoroquinolone antibiotic and glucocorticoids, which were given for endoscopically suspected Crohn's colitis. During the course of the disease no cause of colitis was found other than C. jejuni. Despite the extreme dilatation, the patient was treated conservatively with parenteral nutrition and repeated decompression colonoscopies and made a full, though slow, and uneventful recovery. Follow-up colonoscopies for up to 4 years showed persistent scarring of the transverse colon, probably due to the extreme dilatation, and mild unspecific inflammation of the terminal ileum without histological evidence of inflammatory bowel disease. A comparison with the 6 previously published cases leads to the following conclusions: in most cases the transverse colon is most severely affected. Treatment with either antimotility agents or systemic glucocorticoids does not seem to promote colonic dilatation. The complication has affected patients of both sexes (4 women, 3 men), in the age range of 21 to 83 years, most of them without an underlying disease. The interval between the start of diarrhea and development of the megacolon ranged widely from 3 to 33 days, as did recovery time (2 days to several months). Three of the 7 patients underwent colectomy for imminent or actual colonic perforation. The delayed recovery of our patient was partly attributed to colonic damage caused by extreme dilatation, leading to ischaemia and subsequent scarring of the mucosa, which persisted. Histologically no Crohn's disease or ulcerative colitis could be found at any stage. A rapid increase in resistance of C. species against fluoroquinolone antibodies has been observed in recent years, due to use of the antibiotics in farming. Our patient's severe illness may partly have resulted from delayed effective antibiotic treatment due to resistance. Antibiotic resistance to common enteropathogens should be considered in the case of unusually prolonged or severe enterocolitis. The level of suspicion for either infection or inflammatory bowel disease should remain high as it may be impossible to distinguish between them on the basis of clinical or endoscopic criteria alone.     

Molecular characterization of epididymal proteins

Reduction in energy consumption is known to inhibit development of a variety of spontaneous, carcinogen-induced, and hormone-dependent cancers, but the mechanism or mechanisms by which this occurs remain unknown. We hypothesize that energy consumption may modulate development of estrogen-dependent neoplasms by altering the manner in which target cells respond to estrogens. To test this hypothesis, ovariectomized female Fischer 344 rats were fed diets that allowed consumption of different amounts of energy, and the ability of 17beta-estradiol (E2), administered for 10 wk from subcutaneous Silastic implants, to promote development of prolactin-producing pituitary tumors was examined. A 40% restriction of energy consumption virtually abolished the ability of E2 to promote development of pituitary tumors and associated hyperprolactinemia. A 25% restriction of energy consumption appeared to slightly inhibit E2-induced pituitary growth and hyperprolactinemia, but the observed degree of inhibition was not statistically significant. Interestingly, dietary energy restriction did not inhibit induction by E2 of pituitary cell proliferation and lactotroph hyperplasia. Furthermore, E2 treatment inhibited expression of testosterone-repressed prostate message-2 mRNA, a cellular marker of apoptosis, and this inhibitory effect of E2 was blocked by 40% energy restriction. These data suggest that dietary energy restriction virtually abolished E2-induced development of prolactin-producing pituitary tumors, not by blocking the ability of E2 to induce cell proliferation but rather by blocking the ability of E2 to enhance cell survival. This study and the accompanying paper provide the first indication that dietary energy consumption may modulate estrogen action at the level of the target cell.     

Fatal Campylobacter jejuni infection in a patient splenectomised for thalassaemia

A 35 year old man with a fatal Campylobacter jejuni infection is described. He had HbE/beta zero thalassaemia and had undergone splenectomy nine months previously for hypersplenism; he also had chronic hepatitis C infection. He presented with high grade fever but no gastrointestinal symptoms and rapidly progressed to septicaemic shock and hepatic encephalopathy despite treatment with penicillin, gentamicin, and, later, chloramphenicol and ceftazidime. Only one case of Campylobacter jejuni septicaemia occurring post-splenectomy has been reported previously, also in an iron overloaded thalassaemia patient. Unusual Gram negative bacilli must be covered by the chosen antibiotic regimen when splenectomised thalassaemic patients present with high grade fever.     

Susceptibility of Campylobacter fetus subsp. jejuni to twenty-nine antimicrobial agents

The activity of 29 antimicrobial agents was tested against 95 strains of Campylobacter fetus subsp. jejuni isolated from human stools. Furazolidone and gentamycin were the most active agents. The tetracyclines, erythromycin, and chloramphenicol were very active against most of the strains, but with each antibiotic a few resistant strains were found. The penicillins were relatively inactive, and the cephalosporins tested were only active against occasional strains.     

Evaluation of antibodies reactive with Campylobacter jejuni in Egyptian diarrhea patients

Penis calcinosis is a rare pathology and only two previous cases have been reported in literature. We describe the clinicopathologic features of a case of nodular foreskin calcinosis in a 25-year-old man. The patient's history resulted negative for local trauma, inflammatory disorders or metabolic diseases. The mass measured up to 2 cm and was histologically constituted by multiple intradermic calcium deposits, whose deepest ones were surrounded by epithelioid histiocytes and multinucleated giant cells, with no evidence of any epithelial structures around none of them. These features were consistent with a non-metastatic calcinosis, likely idiopathic, even though also dystrophic calcinosis, observed at its end-stage, may show the same microscopic aspect. The exact idiopathic/dystrophic nosology is briefly discussed.     

Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

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 lambda 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 degreesC 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.     

RAPD typing of Campylobacter jejuni and comparison with Lior's or Penner's serotyping system

Campylobacter jejuni were isolated from 7 epidemic outbreaks (121 isolates), 15 patients with gastroenteritis, chicken meats (47 isolates) and chicken cecal contents (70 isolates). The isolates and one standard strain of C. jejuni JCM2013 were analysed by randomly amplified polymorphic DNA method (RAPD). Total of 254 C. jejuni isolates were divided 68 different RAPD types which included strains that did not to divided by Lior's or Penner's serotyping system. To compare the similarities of RAPD patterns among the isolates, the amplification patterns of DNA were estimated by means of the Dice coefficient, and clustering of strains was based on the unweighted average pair group method (UPGMA) to facilitate the plotting of a dendrogram. It suggests that amplification band patterns of human isolates were different from those of chicken ones. Thus additional information given from RAPD profiles serves for epidemiological investigation and RAPD analysis is recommended as rapid and effective typing method.     

Antimicrobial resistance of clinical strains of Campylobacter jejuni subsp. jejuni isolated from 1985 to 1997 in Quebec, Canada

The antimicrobial resistance of 158 Campylobacter jejuni strains isolated from humans in Quebec, Canada, from 1995 to 1997 was compared to the resistance of 47 and 86 strains of C. jejuni isolated in 1985 and 1986 and in 1992 and 1993, respectively. Of the 291 C. jejuni strains tested, no strain was resistant to erythromycin. Compared to the C. jejuni strains isolated in 1985 and 1986, the C. jejuni strains isolated in 1992 and 1993 were more resistant to tetracycline (40.7 versus 19.1%, respectively; P = 0. 01) but not to nalidixic acid or ciprofloxacin (P > 0.05). Compared to the C. jejuni strains isolated in 1992 and 1993 and in 1985 and 1986, the C. jejuni strains isolated from 1995 to 1997 were more resistant to tetracycline (55.7% versus 40.7 and 19.1%, respectively; P = 0.03 and P < 0.001, respectively) to nalidixic acid (13.9% versus 4.7 and 0%, respectively; P = 0.02 and P = 0.007, respectively), and to ciprofloxacin (12.7% versus 3.5 and 0%, respectively; P = 0.02 and P = 0.009, respectively).     

Evidence of genomic instability in Campylobacter jejuni isolated from poultry

Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with SmaI were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.     

Campylobacter jejuni cytolethal distending toxin causes a G2-phase cell cycle block

Cytolethal distending toxin (CDT) from the diarrheagenic bacterium Campylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease by C. jejuni and other diarrheagenic bacteria that produce CDT.