Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.     

Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides.     

Reducing Campylobacter jejuni Colonization of Poultry via Vaccination

Campylobacter jejuni is a leading bacterial cause of human gastrointestinal disease worldwide. While C. jejuni is a commensal organism in chickens, case-studies have demonstrated a link between infection with C. jejuni and the consumption of foods that have been cross-contaminated with raw or undercooked poultry. We hypothesized that vaccination of chickens with C. jejuni surface-exposed colonization proteins (SECPs) would reduce the ability of C. jejuni to colonize chickens, thereby reducing the contamination of poultry products at the retail level and potentially providing a safer food product for consumers. To test our hypothesis, we injected chickens with recombinant C. jejuni peptides from CadF, FlaA, FlpA, CmeC, and a CadF-FlaA-FlpA fusion protein. Seven days following challenge, chickens were necropsied and cecal contents were serially diluted and plated to determine the number of C. jejuni per gram of material. The sera from the chickens were also analyzed to determine the concentration and specificity of antibodies reactive against the C. jejuni SECPs. Vaccination of chickens with the CadF, FlaA, and FlpA peptides resulted in a reduction in the number of C. jejuni in the ceca compared to the non-vaccinated C. jejuni-challenged group. The greatest reduction in C. jejuni colonization was observed in chickens injected with the FlaA, FlpA, or CadF-FlaA-FlpA fusion proteins. Vaccination of chickens with different SECPs resulted in the production of C. jejuni-specific IgY antibodies. In summary, we show that the vaccination of poultry with individual C. jejuni SECPs or a combination of SECPs provides protection of chickens from C. jejuni colonization.     

Respiratory physiology and energy conservation efficiency of Campylobacter jejuni

A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.     

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.     

Evaluation of antigenic properties of Campylobacter jejuni and Campylobacter coli proteins in a western-immunoblot

Campylobacter jejuni and Campylobacter coli are the most common bacterial cause for acute diarrheal illnesses in developed countries. The aim of this study was to evaluate the antigenic properties of Campylobacterjejuni and Campylobacter coli proteins in western-blot assay. Whole-cell components of Campulobacter jejuni and Campylobacter coli were separated by sodium dodecyl sulfate-polyacrylamide gel electroforesis. Using this method we detected in all seven C. jejuni strains 21 peptides migrating between 180-29 kDa. All three Ccoli strains had a 17 bands migrating with the same molecular weight range. Proteins were transferred electrophoretically to nitrocellulose paper for immunoblotting experiments. The 74 kDa protein reacted strongly in all classes ofimmmunoglobulin with all tested human serum samples. We observed that this protein reacted also with human immunoglobulins for Salmonella and Yersinia sp. This cross-reaction observed for this protein could give false positive results in routine diagnosis of C. jejuni infections. The proteins with molecular weight of: 92, 62, 56, 52, 45-43, 29 kDa were most recognized in the 20 human serum samples. The other proteins of Cljejuni and C. coli, particularly in the 68-50 kDa and 45-31 kDa regions, were recognized occasionally and the response to these in reconvalescent sera was usually weak. The result of this study showed that the proteins with molecular weight: 92, 62, 56, 52, 45-43 and 29 kDa can be use in routine serological diagnostic of campylobacteriosis.     

Bordetella effector BopN is translocated into host cells via its N‐terminal residues

Bordetella bronchiseptica infects a wide variety of mammals, and the type III secretion system (T3SS) is involved in long‐term colonization by Bordetella in the trachea and lung. T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alterations in the host's physiological function. The Bordetella effectors BopN and BteA are known to have roles in up‐regulation of IL‐10 and cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of the BopN translocation into host cells, we built truncated derivatives of BopN and evaluated the derivatives’ ability to translocation into host cells by adenylate cyclase‐mediated translocation assay. It was found that N‐terminal amino acid (aa) residues 1–200 of BopN are sufficient for its translocation into host cells. Interestingly, BopN translocation was completely blocked by deletion of the N‐terminal aa residues 6–50, indicating that the N‐terminal region is critical for BopN translocation. Furthermore, BopN appears to play an auxiliary role in BteA‐mediated cytotoxicity. Thus, BopN can apparently translocate into host cells and may facilitate activity of BteA.

     

L‐fucose influences chemotaxis and biofilm formation in Campylobacter jejuni

Campylobacter jejuni and Campylobacter coli are zoonotic pathogens once considered asaccharolytic, but are now known to encode pathways for glucose and fucose uptake/metabolism. For C. jejuni, strains with the fuc locus possess a competitive advantage in animal colonization models. We demonstrate that this locus is present in > 50% of genome‐sequenced strains and is prevalent in livestock‐associated isolates of both species. To better understand how these campylobacters sense nutrient availability, we examined biofilm formation and chemotaxis to fucose. C. jejuni NCTC11168 forms less biofilms in the presence of fucose, although its fucose permease mutant (fucP) shows no change. In a newly developed chemotaxis assay, both wild‐type and the fucP mutant are chemotactic towards fucose. C. jejuni 81‐176 naturally lacks the fuc locus and is unable to swim towards fucose. Transfer of the NCTC11168 locus into 81‐176 activated fucose uptake and chemotaxis. Fucose chemotaxis also correlated with possession of the pathway for C. jejuni RM1221 (fuc+) and 81116 (fuc‐). Systematic mutation of the NCTC11168 locus revealed that Cj0485 is necessary for fucose metabolism and chemotaxis. This study suggests that components for fucose chemotaxis are encoded within the fuc locus, but downstream signals only in fuc + strains, are involved in coordinating fucose availability with biofilm development.     

The identification of outer membrane proteins and flagella of Campylobacter jejuni

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.     

Utilization of host‐derived cysteine‐containing peptides overcomes the restricted sulphur metabolism of Campylobacter jejuni

The non‐glycolytic food‐borne pathogen Campylobacter jejuni successfully colonizes the intestine of various hosts in spite of its restricted metabolic properties. While several amino acids are known to be used by C. jejuni as energy sources, none of these have been found to be essential for growth. Here we demonstrated through phenotype microarray analysis that cysteine utilization increases the metabolic activity of C. jejuni. Furthermore, cysteine was crucial for its growth as C. jejuni was unable to synthesize it from sulphate or methionine. Our study showed that C. jejuni compensates this limited anabolic capacity by utilizing sulphide, thiosulphate, glutathione and the dipeptides γGlu–Cys, Cys–Gly and Gly–Cys as sulphur sources and cysteine precursors. A panel of C. jejuni mutants in putative peptidases and peptide transporters were generated and tested for their participation in the catabolism of the cysteine‐containing peptides, and the predicted transporter protein CJJ81176_0236 was discovered to facilitate the growth with the dipeptide Cys–Gly, Ile–Arg and Ile–Trp. It was named Campylobacter peptide transporter A (CptA) and is the first representative of the oligopeptide transporter OPT family demonstrated to participate in the glutathione‐derivative Cys–Gly catabolism in prokaryotes. Our study provides new insights into how host‐ and microbiota‐derived substrates like sulphide, thiosulphate and short peptides are used by C. jejuni to compensate its restricted metabolic capacities.     

Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies

Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.     

Immobilization of proteins via arginine residues

A new method for activating polyacrylamide beads to bind proteins via arginine residues is described. The linking reagent, 4-(oxyacetyl)phenoxyacetic acid (OAPA), has been synthesized and characterized. OAPA reacts with arginine or N alpha-acetyl-L-arginine with a stoichiometry of 2 to 1. As expected for an arginine-specific reagent, OAPA inactivates horse liver alcohol dehydrogenase in a time-dependent manner, with the rate of this inactivation decreasing sixfold in the presence of 1 mM NADH. The presence of the carboxyl group in the linking reagent allows efficient coupling to aminated polyacrylamide beads. These derivatized beads are capable of binding various proteins via arginine residues in a time- and pH-dependent manner. Capacities range from less than 0.5 mg/ml to greater than 11 mg/ml, depending on the protein. The proteins are bound in a stable linkage, and preblocking the beads with either arginine or N alpha-acetyl-L-arginine eliminates all protein binding. Preblocking of the protein ubiquitin with OAPA reduces binding to a level compatible with the amount of underivatized ubiquitin remaining. The specificity, water solubility, negative charge, and linking ability of OAPA make it an especially valuable tool, both as a protein-modification reagent and as a linking reagent in preparing specialized affinity chromatographic media.     

On the microbiological diagnostics of Campylobacter jejuni

Investigation of campylobacteriosis cases in 1983-1989 resulted in the isolation of a total of 245 antigenically identified and 23 unidentified strains from humans, animals and foods. A commonly accepted method developed in 1985 using our own experience was used for strain isolation and culturing. A variety of nutrient media in combination with different supplementary substances (antibiotics, growth factors) and additives, such as horse serum, were verified as well as filtration and Fortner's procedures. The best results were obtained when material was stored in thioglycolate transport medium accompanied by cold enrichment (24 h at 4 degrees C) and repeated inoculation into appropriate solid nutrient medium. Owing to the simple culturing of C. jejuni, the number of not elucidated diarrheas was reduced and the incidence of campylobacteriosis (approximately 12 %) is higher than that of salmonellosis and shigellosis. A total of 245 C. jejuni strains was classified using Kahlich's antigenic scheme. The incidence of serovars 1 and 2 was greater than 10 %. Five serovars (13, 17, 25, 26 and 27) were represented by only one strain. The study of campylobacteriosis also revealed the long-term excretion of C. jejuni by convalescents (71 days at most) as well as the occurrence of family outbreaks. Procedures were developed to ensure short-term and long-term (freeze-drying) preservation of isolated strains. The number of cases reported by microbiological laboratories in the framework of the Hygienic Service throughout Czechoslovakia suggest an increase in positive findings with C. jejuni as the etiological agent.     

Identification of the active site residues in ATP‐citrate lyase's carboxy‐terminal portion

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.     

Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni

Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.     

Simultaneous display of multiple foreign peptides in the FliD capping and FliC filament proteins of the Escherichia coli flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

Self-assembly and type III protein export of the bacterial flagellum

The bacterial flagellum is a supramolecular structure consisting of a basal body, a hook and a filament. Most of the flagellar components are translocated across the cytoplasmic membrane by the flagellar type III protein export apparatus in the vicinity of the flagellar base, diffuse down the narrow channel through the nascent structure and self-assemble at its distal end with the help of a cap structure. Flagellar proteins synthesized in the cytoplasm are targeted to the export apparatus with the help of flagellum-specific chaperones and pushed into the channel by an ATPase, whose activity is controlled by its regulator to enable the energy of ATP hydrolysis to be efficiently coupled to the translocation reaction. The export apparatus switches its substrate specificity by monitoring the state of flagellar assembly in the cell exterior, allowing this huge and complex macromolecular assembly to be built efficiently by a highly ordered and well-regulated assembly process.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.