The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.     

galE基因敲除后空肠弯曲菌变异株脂寡糖结构的改变

目的：研究galE基因敲除后空肠弯曲菌变异株脂寡糖结构改变。方法：提取空肠弯曲菌(CJ）HB9313及galE^-变异株脂寡糖，Tricine-SDS聚丙烯酰胺凝胶电泳分离后，分别进行银染、免疫印迹及霍乱毒素配体印迹检查。结果：与野生株比较变异株的脂寡糖分子量变小，电泳迁移速度快，不能与CJHB9313脂寡糖抗体结合，并失去与其配体结合的能力。结论：galE基因敲除变异株丧失了脂寡糖外核糖基及神经节苷脂CM1样结构，可能成为安全减毒活菌苗的有力候选者。     

Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence

This work aimed to establish the role of gne (encoding UDP-GalNAc 4-epimerase activity) and galE (encoding UDP-Gal-4-epimerase activity) in the biosynthesis of surface polysaccharides, as well as in the virulence for eels and humans of the zoonotic serovar of Vibrio vulnificus biotype 2, serovar E. DNA sequence data revealed that gne and galE are quite homologous within this species (> or =90% homology). Mutation in gne of strain CECT4999 increased the surface hydrophobicity, produced deep alterations in the outer membrane architecture, and resulted in noticeable increases in the sensitivity to microcidal peptides (MP), to eel and human sera, and to phagocytosis/opsonophagocytosis. Furthermore, significant attenuation of virulence for eels and mice was observed. By contrast, mutation in galE did not alter the cellular surface, did not increase the sensitivity to MP, serum, or phagocytosis, and did not affect the virulence for fish and mice. The change in the attenuated-virulence phenotype produced by a mutation in gne was correlated with the loss of the O-antigen lipopolysaccharide (LPS), while the capsule was maintained. Complementation of a gne-deficient mutant restored the LPS structure together with the whole virulence phenotype. In conclusion, gne, but not galE, is essential for LPS biosynthesis and virulence in the zoonotic serovar of V. vulnificus biotype 2.     

Cj1136 is required for lipooligosaccharide biosynthesis, hyperinvasion, and chick colonization by Campylobacter jejuni

Campylobacter jejuni is a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasive C. jejuni strains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue of cj1136, which encodes a putative galactosyltransferase according to the annotation of the C. jejuni NCTC11168 genome. In the current study, we investigated the role of cj1136 in C. jejuni virulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. The cj1136 mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation of cj1136 resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. The cj1136 mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded by cj1136 is involved in LOS biosynthesis and is important for C. jejuni virulence, as disruption of this gene and the resultant truncation of LOS affect both colonization in vivo and invasiveness in vitro.     

Critical role of LuxS in the virulence of Campylobacter jejuni in a guinea pig model of abortion

Previous studies on Campylobacter jejuni have demonstrated the role of LuxS in motility, cytolethal distending toxin production, agglutination, and intestinal colonization; however, its direct involvement in virulence has not been reported. In this study, we demonstrate a direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxS complement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902 luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxS gene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivo model of natural disease.     

Lipopolysaccharide structures in Enterobacteriaceae, Pseudomonas aeruginosa, and Vibrio cholerae are immunologically related to Campylobacter spp.

To determine whether lipopolysaccharide (LPS) structures of Campylobacter species are immunologically related to those of 11 other gram-negative organisms, we immunoblotted from polyacrylamide gels the LPS of these strains with immune rabbit serum raised against six Campylobacter jejuni strains and two Campylobacter fetus strains. The LPS studied were from Salmonella minnesota wild type and Ra to Re mutants, Salmonella typhi, Escherichia coli, Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa. None of the 11 LPS preparations was recognized by the eight antisera, but antisera to each of the Campylobacter strains recognized core determinants of some LPS preparations. Antiserum directed against the most serum-sensitive C. jejuni strain, 79-193, was the only antiserum sample that recognized core regions of the rough Salmonella mutants. In converse experiments, when LPS preparations from five Campylobacter strains were blotted with antiserum to Salmonella lipid A, recognition of core structures of each was shown; data from an enzyme-linked immunosorbent assay confirmed this result. In contrast, antiserum to Salmonella typhimurium Re LPS showed no reactivity. We conclude that LPS of Campylobacter strains share lipid A antigenic determinants with the core region of LPS of several other gram-negative organisms.     

Role of catalase in Campylobacter jejuni intracellular survival

The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.     

Some galE mutants of Salmonella choleraesuis retain virulence.

galE mutants were isolated from three mouse-virulent strains of Salmonella choleraesuis (of group C1, O antigen 6,7) by selection for resistance to 2-deoxygalactose. The galE derivative of strain 381 comprised two components: galactose sensitive, thought to be the original mutant; and galactose resistant, presumably by a second mutation reducing galK or galT function or both. The galactose-sensitive component had an intraperitoneal 50% lethal dose for BALB/c mice of ca. 4 X 10(6) CFU, whereas the galactose-resistant component was about as virulent as its gal+ parent, with a 50% lethal dose of ca. 100 CFU. The galE mutant of strain 110 was somewhat sensitive to galactose, as shown by retardation of growth; its 50% lethal dose, ca. 500 CFU, was not much greater than the ca. 200 CFU value for its parent. The galE mutant of strain 117 showed the same partial sensitivity to galactose as strain 110 galE, but was nonvirulent (50% lethal dose of ca. 10(6) CFU versus ca. 400 CFU for its parent). Growth on galactose-supplemented medium restored the smooth phenotype, as indicated by phage sensitivity to three of the four galE strains, but only partially so for the strain 117 galE mutant. The retention of parental virulence by galE mutants of S. choleraesuis which are galactose resistant or somewhat galactose sensitive contrasts with the greatly reduced virulence of galactose-resistant galE mutants of Salmonella typhimurium and Salmonella typhi; this difference may result from the absence of galactose from the O repeat unit in the lipopolysaccharide of group C1 salmonellae.     

Porphyromonas gingivalis galE is involved in lipopolysaccharide O-antigen synthesis and biofilm formation

Porphyromonas gingivalis is a crucial component of complex plaque biofilms that form in the oral cavity, resulting in the progression of periodontal disease. To elucidate the mechanism of periodontal biofilm formation, we analyzed the involvement of several genes related to the synthesis of polysaccharides in P. gingivalis. Gene knockout P. gingivalis mutants were constructed by insertion of an ermF-ermAM cassette; among these mutants, the galE mutant showed some characteristic phenotypes involved in the loss of GalE activity. As expected, the galE mutant accumulated intracellular carbohydrates in the presence of 0.1% galactose and did not grow in the presence of galactose at a concentration greater than 1%, in contrast to the parental strain. Lipopolysaccharide (LPS) analysis indicated that the length of the O-antigen chain of the galE mutant was shorter than that of the wild type. It was also demonstrated that biofilms generated by the galE mutant had an intensity 4.5-fold greater than those of the wild type. Further, the galE mutant was found to be significantly susceptible to some antibiotics in comparison with the wild type. In addition, complementation of the galE mutation led to a partial recovery of the parental phenotypes. We concluded that the galE gene plays a pivotal role in the modification of LPS O antigen and biofilm formation in P. gingivalis and considered that our findings of a relationship between the function of the P. gingivalis galE gene and virulence phenotypes such as biofilm formation may provide clues for understanding the mechanism of pathogenicity in periodontal disease.     

Fitness cost of fluoroquinolone resistance in Campylobacter coli and Campylobacter jejuni

In this study, the fitness cost of fluoroquinolone resistance was evaluated in vitro, on food matrices, and in vivo, using Campylobacter coli and Campylobacter jejuni in vitro selected mutants. In vitro, the growth rate of the susceptible (wild type) and resistant (mutant) strains did not differ when cultured separately. However, by conducting sequential passages of mixed cultures, the ratio of the resistant mutant to the susceptible strain decreased for C. coli but not for C. jejuni. When the wild type and the mutant were co-inoculated on food matrices, mutants were no longer detectable 3 to 5 days after artificial contamination, but the wild-type strains remained detectable for over 13 days. In mono-inoculated animals, no difference was observed between wild-type and mutant fecal titers. When co-inoculated into chickens, the susceptible strain outcompeted the resistant mutant for C. coli and for C. jejuni. However, for C. coli, if the resistant strain was already present in animals, it could persist at high titers in the digestive tract even in the presence of the wild-type strain. Together, these findings suggest that, depending on strain and study conditions, fluoroquinolone resistance can impose a fitness cost on Campylobacter.     

Involvement of a plasmid in virulence of Campylobacter jejuni 81-176

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.     

Study of the role of the htrB gene in Salmonella typhimurium virulence.

We have undertaken a study to investigate the contribution of the htrB gene to the virulence of pathogenic Salmonella typhimurium. An htrB::mini-Tn10 mutation from Escherichia coli was transferred by transduction to the mouse-virulent strain S. typhimurium SL1344 to create an htrB mutant. The S. typhimurium htrB mutant was inoculated into mice and found to be severely limited in its ability to colonize organs of the lymphatic system and to cause systemic disease in mice. A variety of experiments were performed to determine the possible reasons for this loss of virulence. Serum killing assays revealed that the S. typhimurium htrB mutant was as resistant to killing by complement as the wild-type strain. However, macrophage survival assays revealed that the S. typhimurium htrB mutant was more sensitive to the intracellular environment of murine macrophages than the wild-type strain. In addition, the bioactivity of the lipopolysaccharide (LPS) of the htrB mutant was reduced compared to that of the LPS from the parent strain as measured by both a Limulus amoebocyte lysate endotoxin quantitation assay and a tumor necrosis factor alpha bioassay. These results indicate that the htrB gene plays a role in the virulence of S. typhimurium.     

Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation

Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.     

Outer membrane characteristics of Campylobacter jejuni.

Outer membranes were isolated from type strains and wild-type isolates of Campylobacter jejuni and Campylobacter coli by sodium lauryl sarcosinate extraction, and the polypeptide complement and lipopolysaccharide (LPS) content were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles exhibited by membranes from both species were quite similar, but could be distinguished from the type strain of the genus, C. fetus subsp. fetus CIP5396. The sodium dodecyl sulfate electrophoretograms of C. jejuni and C. coli were dominated by a major polypeptide band. In the reference strain C. jejuni VC74, this polypeptide had an apparent molecular weight of 45,000, was heat modifiable, and was shown to be transmembrane by virtue of its peptidoglycan association and surface exposure. Two other proteins with approximate molecular weights of 37,000 and 73,000 were also surface exposed on C. jejuni VC74 and represented potential surface antigens. The LPS of C. jejuni and C. coli was of low molecular weight, suggesting that serotypic differences due to LPS were based on different carbohydrate compositions of core LPS. In contrast, the LPS of C. fetus CIP5396 exhibited O antigen polysaccharide chains of intermediate chain length. Fragments of outer membranes released during growth of C. jejuni VC74 displayed a polypeptide profile which differed from that of sarcosinate-extracted outer membranes. Radiolabeling demonstrated that the proteins exposed on the surface of this released membrane differed from those exposed on the cell surface and would likely contribute to the antigenic complexity of C. jejuni.     

Gamma-glutamyl transpeptidase has a role in the persistent colonization of the avian gut by Campylobacter jejuni

The contribution of gamma-glutamyl transpeptidase (GGT) to Campylobacter jejuni virulence and colonization of the avian gut has been investigated. The presence of the ggt gene in C. jejuni strains directly correlated with the expression of GGT activity as measured by cleavage and transfer of the gamma-glutamyl moiety. Inactivation of the monocistronic ggt gene in C. jejuni strain 81116 resulted in isogenic mutants with undetectable GGT activity; nevertheless, these mutants grew normally in vitro. However, the mutants had increased motility, a 5.4-fold higher invasion efficiency into INT407 cells in vitro and increased resistance to hydrogen peroxide stress. Moreover, the apoptosis-inducing activity of the ggt mutant was significantly lower than that of the parental strain. In vivo studies showed that, although GGT activity was not required for initial colonization of 1-day-old chicks, the enzyme was required for persistent colonization of the avian gut.     

The effect of campylobacter lipopolysaccharide on fetal development in the mouse

Purified lipopolysaccharide (LPS) obtained from isolates of Campylobacter fetus ss. fetus and Campylobacter jejuni impaired fetal development when administered to mice on day 13 of pregnancy. Strikingly more fetal resorption was produced by C. jejuni LPS than by similar amounts of C. fetus ss. fetus LPS. Three of the four Campylobacter strains examined produced LPS that had no effect on maternal health, but LPS from one C. jejuni strain killed all of the mice to which it was administered.