The pathogenic potential of Campylobacter concisus strains associated with chronic intestinal diseases

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, "membrane ruffling", cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients.     

Sequencing and validation of the genome of a Campylobacter concisus reveals intra-species diversity

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Its role in different diseases remains a subject of debate; this may be due to strain to strain genetic variation. Here, we sequence and analyze the genome of a C. concisus from a biopsy of a child with Crohn's disease (UNSWCD); the second such genome for this species. A 1.8 Mb genome was assembled with paired-end reads from a next-generation sequencer. This genome is smaller than the 2.1 Mb C. concisus reference BAA-1457. While 1593 genes were conserved across UNSWCD and BAA-1457, 138 genes from UNSWCD and 281 from BAA-1457 were unique when compared against the other. To further validate the genome assembly and annotation, comprehensive shotgun proteomics was performed. This confirmed 78% of open reading frames in UNSWCD and, importantly, provided evidence of expression for 217 proteins previously defined as 'hypothetical' in Campylobacter. Substantial functional differences were observed between the UNSWCD and the reference strain. Enrichment analysis revealed differences in membrane proteins, response to stimulus, molecular transport and electron carriers. Synteny maps for the 281 genes not present in UNSWCD identified seven functionally associated gene clusters. These included one associated with the CRISPR family and another which encoded multiple restriction endonucleases; these genes are all involved in resistance to phage attack. Many of the observed differences are consistent with UNSWCD having adapted to greater surface interaction with host cells, as opposed to BAA-1457 which may prefer a free-living environment.     

Molecular identities of human sperm proteins reactive with antibodies in sera of immunoinfertile women

Antisperm antibodies (ASA) can cause infertility in both men and women. It is important to delineate the sperm antigens against which these ASA are directed. Sperm proteins were separated by 2D gel electrophoresis and transferred to nitrocellulose membrane and incubated with sera from fertile women or immunoinfertile women having ASA. The corresponding immunoreactive peptide spots were cored from the gel and analyzed by the two-dimensional (2D) gel electrophoresis/matrix-assisted laser desoprtion ionization-time of flight-mass spectrometry and liquid chromatography-mass spectrometry (MALDI-TOF-MS/LC-MS). A total of 68 spots belonging to 38 different proteins and their isomers were identified. Fourteen of these proteins and their isomers reacted with both the fertile and immunoinfertile sera. Twenty-four of these proteins reacted specifically only with the immunoinfertile sera and not with the fertile sera. Among them was a novel protein designated as a hypothetical protein FLJ32704 (accession # Q96MA6). An immunodominant sequence (amino acid 151-159) of this protein was identified and a nonamer peptide based upon this sequence (IQTLG1TPR) was synthesized and examined for its immunoreactivity. This synthetic peptide reacted with 90% (36/40) of immunoinfertile sera and not with any of the fertile sera (0/40) in the enzyme-linked immnosorbent assay (ELISA). In conclusion, using the 2D gel electrophoresis/MALDI-TOF-MS/LC-MS procedure, we have identified several known and at least one novel antigen against which the antibodies are present in sera of immunoinfertile but not fertile women. Some of these antigens may find applications in specific diagonsis and treatment of infertility/immunoinfertility, and in the development of new generation of contraceptive modalities including contraceptive vaccines.     

Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli

We have identified several immunoreactive epitopes on the human immunodeficiency virus (HIV) type 1 transmembrane envelope protein by synthesizing various regions of the protein as fusions to the trpE gene in Escherichia coli. Ten fusion clones which expressed overlapping peptides were found to contain epitopes reactive with antibodies in sera of North American (NAm) and West African (WAf) patients with acquired immune deficiency syndrome (AIDS). An immunodominant epitope which reacted with all HIV-infected patients' sera was mapped to a 51-amino acid sequence in the N terminus of p41. A novel epitope was also identified in the C terminus of p41 which was reactive with 41% and 48% of the sera tested from NAm and WAf, respectively. In addition, several minor epitopes were identified. We observed that sera from WAf reacted more strongly to minor HIV-1 p41 epitopes than did sera from similarly infected individuals in NAm. We also report on the detection of antibodies from patients with HIV-2 infection in WAf which cross react with HIV-1 p41 recombinant envelope antigens.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Fine specificities of autoantibodies directed against the Ro, La, Sm, RNP, and Jo-1 proteins defined by two-dimensional gel electrophoresis and immunoblotting

Although useful for specific purposes, immunofluorescence, precipitation in agarose gels, and the m.w. estimation of RNA or proteins immunoprecipitated from transformed cells often provide partial or ambiguous definition of autoantibody specificity. We have analyzed organ and cell extracts by one-and two-dimensional electrophoresis together with Western blotting to define the fine specificities of antibodies to the ribonucleoprotein (RNP) antigens Ro, La, Sm, RNP and Jo-1. One-dimensional analysis identified the Ro protein as a 57 kilodalton (kd) protein, although many anti-Ro sera also react with a 50 kd protein. La antisera react with 50 and 43 kd proteins. The 50 kd La protein readily breaks down into 43, 25, and smaller immunoreactive cleavage products. Partial proteolysis of Ro and La proteins in human spleen extracts produces similar immunoreactive products, providing evidence for a common structure. The major immunoreactive Sm antigens defined by human polyclonal antisera and a mouse monoclonal antiserum were doublets of 25/26 and 16/18 kd, whereas anti-RNP sera reacted with a protein of 68 kd. Most Sm-RNP antisera contained antibodies reactive with additional proteins, especially when whole cell extracts were used as a source of antigens. Two-dimensional analysis provided characteristic maps of the antigens. Ro and La were acidic, and La showed a unique set of acidic charge isomers at 50 and 43 kd. Anti-Sm antibodies reacted with discrete dots corresponding to both the acidic and basic regions of the first-dimension (charge) gels, whereas the RNP antigen showed a series of basic charge isomers of 68 kd. Many anti-Sm-RNP sera reacted with other closely spaced proteins of a similar charge and size to the Sm and RNP antigens, suggesting antibody cross-reactivity or reactivity with closely related functional proteins. Although Jo-1 had the same m.w. as the undegraded La antigen, the fingerprints were quite distinctive on two-dimensional electrophoresis. The results of this study indicate how the source and preparation of antigen extracts, as well as protein degradation, influence the m.w. determinations of soluble protein antigens. With these factors taken into account, two-dimensional fractionation with immunoblotting provides a highly discriminating, sensitive, and reproducible method of analysis of autoantibody specificity. This technique can be used to standardize reference antisera and to study protein antigens in normal and abnormal cell and tissue extracts, and could lead to new or more precise correlations with clinical disease.     

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.     

Characterization of Mycobacterium paratuberculosis p36 Antigen and Its Seroreactivities in Crohn's Disease

Recent data using improved cultural, molecular, and serological techniques have strengthened the association of Mycobacterium paratuberculosis with Crohn's disease, an inflammatory bowel disease (IBD) with unknown etiology. To provide more evidence of an etiological association, antibody reactivities of Crohn's disease patients were tested by immunoblotting against M. paratuberculosis–recombinant antigens. A clone containing a 1,402-bp insert and expressing a 36K-antigen (p36) was analyzed. No homology was found between the deduced amino acid sequence of p36 and any protein sequences compiled in the GenBank indicating that p36 is a novel mycobacterial protein. The reactivity of 199 serum samples was tested against the p36 by immunoblotting technique. Sera from 77 of 89 (86.5%) Crohn's disease patients and 16 of 18 (89%) sera from patients with tuberculosis and leprosy reacted with p36 compared to 5 of 42 (12%) ulcerative colitis and non-IBD control sera (p < 0.0001). In addition, p36 reacted to all sera from 10 normal controls that were Bacillus Calmette-Guerin (BCG)-immunized and only to 10% of 40 normal controls that were not BCG-immunized. The fact that sera from Crohn's disease patients reacted to p36 with the same high frequency as the sera from patients that were exposed to mycobacterial antigens further supports the hypothesis of the mycobacterial etiology in Crohn's disease. Received: 8 January 1998 / Accepted: 18 March 1999     

Proteomic analysis of anti-Francisella tularensis LVS antibody response in murine model of tularemia

Francisella tularensis live vaccine strain infection of mice has been established as an experimental model of tularemia that is suitable for studies of immune mechanisms against the intracellular pathogen. In this study, the model was used to explore immunogenic repertoire of F. tularensis with the aim of identifying new molecules able to activate the host immune system, potential bacterial markers with vaccine, and diagnostic applications. Immunoproteomic approach based on the combination of two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry was applied. Globally, 36 different proteins were identified, which strongly reacted with sera from experimentally infected mice, including several putative virulence markers of intracellular pathogens as nucleoside diphosphate kinase, isocitrate dehydrogenase, RNA-binding protein Hfq, and molecular chaperone ClpB. Of them, 27 proteins are described for the first time as immunorelevant Francisella proteins. When comparing murine immunoproteome of F. tularensis with our previous data from human patients, 25 of the total of 50 identified murine sera immunoreactive spots were recognized by human sera collected from patients suffering from tularemia, as well. Immune sera from two Lps gene congenic strains of mice, C3H/HeN (Lpsn) and C3H/HeJ (Lpsd), represented murine immunoproteome in this study. The spectrum of immunoreactive spots detected by two-dimensional immunoblotting varied throughout the course of infection depending on murine strain. Nevertheless, the antibody patterns of the two strains showed significant homogeneity in being directed against almost identical subset of antigens.     

Serological Screening of the Schistosoma mansoni Adult Worm Proteome

Background

New interventions tools are a priority for schistosomiasis control and elimination, as the disease is still highly prevalent. The identification of proteins associated with active infection and protective immune response may constitute the basis for the development of a successful vaccine and could also indicate new diagnostic candidates. In this context, post-genomic technologies have been progressing, resulting in a more rational discovery of new biomarkers of resistance and antigens for diagnosis.

Methodology/Principal Findings

Two-dimensional electrophoresed Schistosoma mansoni adult worm protein extracts were probed with pooled sera of infected and non-infected (naturally resistant) individuals from a S. mansoni endemic area. A total of 47 different immunoreactive proteins were identified by mass spectrometry. Although the different pooled sera shared most of the immunoreactive protein spots, nine protein spots reacted exclusively with the serum pool of infected individuals, which correspond to annexin, major egg antigen, troponin T, filamin, disulphide-isomerase ER-60 precursor, actin and reticulocalbin. One protein spot, corresponding to eukaryotic translation elongation factor, reacted exclusively with the pooled sera of non-infected individuals living in the endemic area. Western blotting of two selected recombinant proteins, major egg antigen and hemoglobinase, showed a similar recognition pattern of that of the native protein.

Concluding/Significance

Using a serological proteome analysis, a group of antigens related to the different infection status of the endemic area residents was identified and may be related to susceptibility or resistance to infection.     

Characterization of Campylobacter concisus hemolysins

Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight < 10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant.     

Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection

Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C.?pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C.?pneumoniae, we screened 455 genes with unknown function in the genome of C.?pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C.?pneumoniae proteins were subjected to Western blot analysis using serum samples from C.?pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C.?pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C.?pneumoniae infections and for the development of vaccines in future.     

Identification of seroreactive proteins in the culture filtrate antigen of Mycobacterium avium ssp. paratuberculosis human isolates to sera from Crohn's disease patients

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA ( P <0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Disease state differentiation and identification of tuberculosis biomarkers via native antigen array profiling

A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.     

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.     

Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1

Background

Actinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits.

Results

Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge.

Conclusions

We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates.     

Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array

As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged, and stored. NC filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4), and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne's disease. The M. paratuberculosis heat shock protein DnaK, encoded by ORF MAP3840 and a membrane protein (MAP2121c), were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in sera from Johne's disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and has laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Detection of eight antibodies in cancer patients' sera against proteins derived from the adenocarcinoma A549 cell line using proteomics-based analysis

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as alpha-enolase, inosine-5'-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients' sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.