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.     

Identification and molecular cloning of a gene encoding a fibronectin-binding protein (CadF) from Campylobacter jejuni

Campylobacter jejuni , a Gram-negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella , the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni . Ligand immunoblot assays using ¹²⁵I-labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified protein, reacted with a 37 kDa protein in all C. jejuni isolates ( n  = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni -infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37 kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36 872 Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens . Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild-type C. jejuni as judged by ligand-binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.     

Enzyme-linked immunosorbent assay in the diagnosis of campylobacteriosis

Campylobacter jejuni and Campylobacter coli are the bacterial cause of human gastroenteritis commonly reported worldwide. The serodiagnosis of Campylobacter infections is not routinely done in Poland so the aim of this study was to evaluation of ELISA in the diagnosis ofcampylobacteriosis. Serum samples obtained from 145 patients with gastroenteritis were tested by ELISA with 7 different heat-stable antigens of C. jejuni and one of C. coli and by the commercial Virion/Serion ELISA with purified 45 kDa outer membrane protein of C. jejuni. Antibodies for heat-stable antigens of C. jejuni were detected statistically more often than antibodies for heat-stable antigens of C. coli and for purifled protein of C. jejuni. We found significant differences in the frequency of detection of antibodies to different heat-stable antigens, ranged from 18.6% to 68.9% of positive results, what indicate for serological heterogenicity of C. jejuni strains isolated in Poland. The results of our study showed usefulness of ELISA in serological diagnosis of campylobacteriosis. However it is necessary to serotype the C. jejuni strains isolated in Poland to find the appropriate C. jejuni serotype for using in ELISA.     

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.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.     

Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal antibodies

Poultry are considered the major reservoir for Campylobacter jejuni, a leading bacterial cause of human food-borne diarrhea. To understand the ecology of C. jejuni and develop strategies to control C. jejuni infection in the animal reservoir, we initiated studies to examine the potential role of anti-Campylobacter maternal antibodies in protecting young broiler chickens from infection by C. jejuni. Using an enzyme-linked immunosorbent assay (ELISA), the prevalence of anti-C. jejuni antibodies in breeder chickens, egg yolks, and broilers from multiple flocks of different farms were examined. High levels of antibodies to the organism were detected in serum samples of breeder chickens and in egg yolk contents. To determine the dynamics of anti-Campylobacter maternal antibody transferred from yolks to hatchlings, serum samples collected from five broiler flocks at weekly intervals from 1 to 28 or 42 days of age were also examined by ELISA. Sera from the 1-day and 7-day-old chicks showed high titers of antibodies to C. jejuni. Thereafter, antibody titers decreased substantially and were not detected during the third and fourth weeks of age. The disappearance of anti-Campylobacter maternal antibodies during 3 to 4 weeks of age coincides with the appearance of C. jejuni infections observed in many broiler chicken flocks. As shown by immunoblotting, the maternally derived antibodies recognized multiple membrane proteins of C. jejuni ranging from 19 to 107 kDa. Moreover, in vitro serum bactericidal assays showed that anti-Campylobacter maternal antibodies were active in antibody-dependent complement-mediated killing of C. jejuni. Together, these results highlight the widespread presence of functional anti-Campylobacter antibodies in the poultry production system and provide a strong rationale for further investigation of the potential role of anti-C. jejuni maternal antibodies in protecting young chickens from infection by C. jejuni.     

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Proteomic and immunoproteomic analysis of Aggregatibacter actinomycetemcomitans JP2 clone strain HK1651

The proteome of Aggregatibacter actinomycetemcomitans HK1651 (JP2 clone) and immunoreactive antigens were studied by two-dimensional (2D) gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and 2D immunoblotting. The highly leukotoxic JP2 clone of A. actinomycetemcomitans is strongly associated with aggressive periodontitis (AgP) in adolescents of North-West African descent and the pathogenicity of this bacterium is of major interest. Hence, we developed a comprehensive 2D proteome reference map of A. actinomycetemcomitans proteins with 167 identified spots representing 114 different proteins of which 15 were outer membrane proteins. To unravel immunoreactive antigens, we applied 2D-gel and subsequent immunoblotting analyses using sera from five individuals with A. actinomycetemcomitans infections and one healthy control. The analysis revealed 32 immunoreactive proteins. Antibodies to two outer membrane proteins, YaeT (85 kDa) and Omp39 (39 kDa), not previously described as immunoreactive, were found only in subjects with current or previous A. actinomycetemcomitans JP2 infection. Further proteome-based studies of A. actinomycetemcomitans combined with analyses of the humoral immune response and targeted against outer membrane proteins may provide important insight into the host relationship of this important pathogen.     

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes

Campylobacter jejuni is one of the major causes of human diarrhea throughout the world. Attachment to host cells and extracellular matrix proteins is considered to be an essential primary event in the pathogenesis of enteritis. Outer membrane proteins of three C. jejuni strains, one of which was aflagellate, were investigated for their contribution to the process of adhesion to INT 407 cell membranes and the extracellular matrix protein fibronectin. Using a ligand-binding immunoblotting assay the flagellin, the major outer membrane protein and a 59-kDa protein were detected to be involved in adhesion to both substrates. The MOMP was able to inhibit the attachment of the bacteria to INT 407 cell membranes partly, when the protein was isolated under native conditions. However, it was totally lost when the protein was isolated in the presence of SDS. The 59-kDa protein of one strain was identified by N-terminal sequencing, and regarding the first 14 amino acids it was found to be identical to the 37-kDa CadF protein just recently described as fibronectin-binding protein of C. jejuni. Especially for the aflagellate strain this protein may be of special importance for adhesion of the bacteria to different substrates.     

Outer membrane proteins of Fusobacterium nucleatum Fev1

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100 degrees C, respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni

It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To characterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), Coomassie-stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild-type strains were analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA-reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA-reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid-glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of alpha-linked N-acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non-flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain alpha-linked N-acetylgalactosamine residues.     

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.     

Genomic differences between Campylobacter jejuni isolates identify surface membrane and flagellar function gene products potentially important for colonizing the chicken intestine

Campylobacter spp. are one of the leading bacterial etiologic agents of acute human gastroenteritis among industrialized countries. Poultry are implicated as a major source of the organism for human illness; however, the factors involved with colonization of poultry gastrointestinal systems remain unclear. Genomics and proteomics analyses were used to identify differences between poor- versus robust-colonizing Campylobacter jejuni isolates, 11168(GS) and A74/C, respectively. Sequence analyses of subtracted DNA resulted in A74/C-specifc genes similar to a dimethyl sulfoxide reductase, a serine protease, polysaccharide modification proteins, and restriction modification proteins. DNA microarray analyses were performed for comparison of A74/C to the complete genome sequences published for two C. jejuni. A total of 114 genes (7.1%) were determined absent from A74/C relative to those genomes. Additionally, proteomics was completed on both soluble and membrane protein extracts from 11168(GS) and A74/C. Variation in protein expression and physical characteristics such as pI was detected between the two isolates that included the major outer membrane protein, flagella, and aconitate hydratase. Several proteins including cysteine synthase and a Ni/Fe hydrogenase were determined to be differentially present between the two isolates. Finally, DNA hybridization analyses of 19 C. jejuni isolates recovered from chickens and humans worldwide over the past 20 years were performed to determine the distribution of a subset of differentially identified gene sequences.     

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.