Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile

Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C. difficile. A gene library of C. difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified. One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C. difficile strains. Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis. Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C. difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.     

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome

Abstract Screening of a Clostridium difficile ψEMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb Hin dIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, β-hydroxybutyryl coenzyme A dehydrogenase and thiolase).     

Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12

Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits. A gene bank of C. difficile DNA in Escherichia coli was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3. Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified. One of these clones lambda tA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A. The size of the lambda tA5 insert DNA was 14.3 kb.     

Cloning of an Outer Membrane Protein Gene from Campylobacter jejuni

An antigen in the outer membrane protein (OMP) fraction of Campylobacter jejuni was identified and characterized. Western blot analysis demonstrated antigenic differences in this protein between two congenic C. jejuni strains. Strain A74/C, which colonizes chickens, expressed the antigen at 34 kDa, while strain A74/O, which poorly colonizes chickens, expressed the antigen at 32 and 34 kDa. A genomic library was constructed in λgt11 with DNA from A74/O and screened with antibody raised against C. jejuni OMPs. A clone that possessed a 1.3-kb insert and expressed an immunoreactive protein fused to β-galactosidase was isolated and purified. DNA sequence analysis revealed the insert contained one open reading frame 864 bases long. The deduced amino acid sequence demonstrated 56.3% similarity with Bacillus steorothermophilus glnH, a glutamine-binding protein, and 54.0% similarity with C. jejuni PEB1, a putative colonization adhesin. Southern hybridization, Northern hybridization, and DNA sequence analyses of the congenic colonizing and noncolonizing strains of C. jejuni failed to distinguish the two strains and revealed only one copy of the gene. Post-translational modification may be an alternate explanation for the antigenic differences seen between the two strains. Received: 15 October 1996 / Accepted: 3 December 1996     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis.

Using antisera (alpha-R and alpha-C7Ag) directed against the conserved Gly-Gly-Met-Pro-epitope of the hsp70 family, a single antigen was identified in the human Babesia divergens Rouen 1987 isolate by Western immunoblotting and immunoprecipitation experiments. This B divergens hsp70 is highly conserved as shown by the analysis of five other geographical B divergens isolates from different hosts (human and bovine). Indirect immunofluorescence assay performed on the asexual intraerythrocytic stages showed that the hsp70 is mainly cytoplasmic and stage-independent. Heat-shock experiments, with 20 min incubation at 40 degrees C followed by a 10 to 50 min shift to 37 degrees C in the presence of [35S]-methionine, led to an increase of two hsp of 85 and 70 kDa while protein synthesis in general decreased within 10 min. Immunoprecipitations of [35S]-methionine radiolabelled proteins with human, ox and gerbil antisera raised against various B divergens isolates, showed the presence of a B divergens 70 kDa protein which was demonstrated to be a hsp70 by coupling immunoblotting assays with alpha-C7Ag serum on the same immunoprecipitated material. During human babesiosis, the B divergens hsp70 appears as an early antigen during the acute phase. These results are in agreement with the use of the B divergens hsp70 as an essential valence antigen in an anti-babesiosis vaccine.     

Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci

Abstract While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a λgt11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci . These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia .     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Protein composition of mesoglea and mesogloeal cells of medusa Aurelia aurita

Protein composition of mesoglea of the scyphomedusa Aurelia aurita was revealed in SDS-PAGE. Some major bands are visible in mesoglea of a mature medusa: 30, 45-47, 85 kDa, three bands between 100-200 kDa, and several bands with molecular weights > 300 kDa. Polyclonal antisera RA45/47 against protein 45 kDa were raised. RA45/47 react with 45-47 kDa protein in mesogleal sample and protein 120 kDa in mesogleal cells on immunoblot. Immunohistochemical analysis of A. aurita histological sections of young and mature medusae showed antigen localization in mesogleal cell granules and in the apical part of ectodermal cells. In mature medusae, the antigen was localized also in elastic fibers. We can conclude that in A. aurita mesogleal cells, along with ectodermal cells, take part in the formation of extracellular matrix of mesoglea.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin

Abstract A gene library of Neisseria meningitidis B15 P1.16 DNA was established in λ Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of bp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae , and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.     

G1 antigen: a cell-surface immunoprotective 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization

Strains of the fish pathogen Enterococcus seriolicida were identified as agglutinating and non-agglutinating, according to their reaction with anti-serum raised against type strain YT-3 (ATCC49156). The non-agglutinating strains are highly pathogenic in contrast to agglutinating strains. A 96 kDa immunoprotective glycoprotein G1 antigen from non-agglutinating Ent. seriolicida strain SS91-014 (N) was purified and characterized. The purification procedure entailed extraction of antigen by glass bead agitation, 80% (NH4)(2)SO4 precipitation, gel filtration and electroelution. An immunofluorescence microscopy study using monoclonal antibody M3A5 raised against G1 antigen revealed that G1 antigen is present only on the cell surface of non-agglutinating strains. Therefore, the G1 antigen of virulent Ent. seriolicida could be a potential candidate for protective vaccine against enterococcosis in fish.     

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.     

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inc lusion membrane protein A ) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.     

Human cell surface antigens coded by genes on chromosome 21

Clones of man-mouse hybrids derived from four different crosses which retained a very limited number of human chromosomes were studied for the expression of human cell surface antigens. In testing a variety of rabbit antisera to human cells and tissues, it was found that an antiserum to Daudi cells recognizes human species-specific antigen(s) on three ‘WA’ clones, all of which carried human chromosome 21. Absorption of the antiserum with any of the clones abolished its activity against all clones, indicating that the antiserum recognized the same antigen(s) on these clones. The antigen(s) was shown to be present on normal human lymphocytes, more on B than on T cells, but apparently absent from erythrocytes. C3H mice, from which the murine parent originated, were immunized with the WA clones carrying human chromosome 21. The resultant antisera reacted with clones carrying chromosome 21 but not with clones which did not retain this chromosome, even though some of these clones possessed many other human chromosomes. The murine antisera reacted with some, but not all, human peripheral blood lymphocytes tested. Absorption studies clearly showed the multiple nature of the antigens recognized by these antisera. Studies on cells of identical twins provided evidence that these antigens are inheritable.     

Antigenic characterization of Sphaerospora dicentrarchi (Myxosporea: Bivalvulida), a parasite from European sea bass Dicentrarchus labrax (Teleostei: Serranidae)

The biochemical composition of Sphaerospora dicentrarchi was studied. Periodate and Proteinase K treatments as well as lectin blots were used to analyse carbohydrate terminals. Zymography was applied to detect proteases. Four polyclonal antisera, raised against S. dicentrarchi (RaSdic), S. testicularis (RaStest), Ceratomyxa labracis (RaClab) and C. sparusaurati (RaCspr), were used in SDS polyacrylamide gel electrophoresis and immunoblot. Bands with molecular weight (MW) between 32 and 130 kDa were detected by electrophoresis. After Proteinase K treatment, apparent digestion of bands heavier than 43 kDa took place. RaSdic and RaStest detected similar bands with MW between 20 and 50 kDa, whereas RaClab and RaCspr recognized bands between 50 and 140 kDa. The 50 kDa band was recognized by all the polyclonal antisera, suggesting that it could correspond to an antigen shared by several myxosporean parasites. Four proteases were observed by zymography. From the 5 lectins assayed, binding was only observed using Con-A, which detected 2 bands of 96 and 78 kDa. Periodate treatment did not produce any effect on the binding of RaSdic and RaStest, but a high decrease of intensity in the antibody binding occurred at a concentration of 10 and 20 mM periodate when RaClab and RaCspr were tested. These results give information on the antigenic composition of S. dicentrarchi which could be useful for further diagnostic or immunoprevention studies.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.