Identification of major antigenic proteins ofPasteurella piscicida

Two different antigenic protein-coding clones (PPA1 and PPA2) were isolated using anti-Pasteurella piscicidarabbit serum from a genomic DNA library ofP. piscicidastrain KP9038. The PPA1 and PPA2 expressed 7 kDa and 45 kDa proteins inEscherichia coli, respectively, and the molecular sizes of these expressed proteins are the same as these of the major antigenic proteins ofP. piscicida. PPA1 encodes a protein of 83 amino acids residues, which is similar to the bacterial lipoprotein. Comparison of the predicted amino acid sequence of the PPA1-encoded 7 kDa protein ofP. piscicidawith previously reported bacterial lipoprotein sequence data revealed that it shares about 40% amino acid sequence identity. PPA2 has two large open reading frame (ORFs). The larger ORF (encoding 452 amino acid residues) encodes a homolog of DegQ protease, and the smaller ORF (371 amino acid residues) encodes a homolog of DegS protease. The antibodies reacted with the larger ORF-encoded 45 kDa DegQ homolog protein. The DegQ and DegS homolog proteins contain an export signal and a serine protease active site. The structural features of the PPA2-coding locus are similar to those of the loci inE. colifor thedegQanddegSserine protease genes. A sequence in the 3′ non-coding region ofVibrio hollisaethermostable hemolysin gene that is highly homologous with a similar located sequence in thePseudomonas putidap-cresol methylhydroxylase gene is also found in the 3′ non-coding region of thedegShomolog gene of the PPA2.     

Expression ofNeisseria meningitidisclass 1 porin as a fusion protein inEscherichia colli: the influence of liposomes and adjuvants on the production of a bactericidal immune reesponse

High level exxpression of meningococcal class 1 protein was achieved inEscherichia coliusing the p-GEMEX-1 vector, in which the protein was expressed in inclusion bodies, (IB), as a fusion with the bacteriophage T7 gene 10 capsid protein. The fusion protein (FP) was engineered with a factor Xa protease site between the gene 10 and class 1 protein, but treatment with the enzyme resulted in cleavage at additional sites within the class 1 protein. Since it was not possible to remove the leader protein, the intact FP provided an alternative antigen for immunization. Antisera raised to FP, solubilized from IB and incorporated into liposomes, generated a subtype-specific response which was weakly bactericidal for meningococci. In order to remove any possible effect ofE. coliLPS present in IB, the FP was further purified by SDS-PAGE and incorporated into liposomes, either alone ofr in combination with the adjuvants monophosphoryl lipin A or muramyl dipeptide. The incorporation of adjuvants in liposomes resulted in stimulation of the overall immune response to FP, but the resulting antisera were not bactericidal. however and effective bactericidal response was obtained with the purest preparation of FP in liposomes, without any additional adjuvants, revealing that attempts to increase further the immunogenicity of such antigens must not be at the expense of interfering with optimal protein folding     

Characterization of bovine septicemic, bovine diarrheal, and human enteroinvasive Escherichia coli that hybridize with K88 and F41 accessory gene probes but do not express these adhesins

Certain DNA probes derived from accessory genes of cloned K88 and F41 determinants hybridize with Escherichia coli strains that express K88 or F41 and with certain other E. coli strains that do not express these antigens. We found that these probes hybridized with human enteroinvasive E. coli, and with bovine E. coli isolates which produced a fatal septicemia in experimentally infected piglets. These strains did not hybridize with probes derived from the structural subunit genes encoding the K88 and F41 antigens. E. coli strains isolated from turkeys with septicemia, Shigella and Salmonella strains did not hybridize to the K88 and F41 accessory gene probes. The K88 and F41 accessory gene probes hybridized with a 200 kb plasmid which is required for invasion by human enteroinvasive E. coli. The K88 and F41 accessory gene homology in the bovine isolates was located on a 150 kb transmissible plasmid but was unrelated to plasmids encoding aerobactin, Vir, or colicin V, which are suspected virulence factors in septicemic E. coli. A common plasmid-encoded antigen was associated with bovine isolates that hybridized with the K88 and F41 accessory gene probes. This included strains which express CS31A, a surface antigen associated with bovine septicemic E. coli, which also hybridized with the K88 and F4 accessory gene probes. The results suggest that the K88 and F41 accessory gene probes hybridized with sequences that may be associated with a common mechanism of pilus expression in distinct groups of E. coli pathogens.     

Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization

Vibrio cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions. The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V. cholerae filamentous phage CTXф. Interestingly, Zot appears to be structurally and functionally related to the gene I product of other filamentous phages and it has been shown to be required for CTXф morphogenesis. In this study we described the cloning of zot in several expression plasmid systems and we examined the subcellular localization of Zot by using affinity purified anti-Zot antibodies. We found that Zot localizes in the V. cholerae cell envelope with M_r45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa). A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected. Both molecules are exposed at the bacterial cell surface. The production of the 33 kDa Zot, that might represent a processing product, was abolished in mutant ZotG59. N-terminal tagged 6xHis-Zot fusion protein retained the capability to reach the outer membrane and the 6xHis tag was not cleaved off during the translocation to the periplasm, whereas the presence of the tag partially blocked the formation of the 33 kDa molecule. Zot secretion and anchorage to the bacterial outer membrane was also observed in E. coli strains expressing Zot, suggesting that the toxin may be directed to the outer membrane via the same pathway in E. coli and V. cholerae. Zot cleavage might be due to a V. cholerae specific protease activity, since the 33 kDa protein was not efficiently produced in E. coli. On the basis of these data and Zot amino acid sequence analysis, we suggest that while the N-terminal part of the molecule is involved in the morphogenesis of CTXф, the C-terminal region might carry the domain(s) responsible for Zot enterotoxic activity.     

Molecular Basis for Antigenic Variation of a Protective Strain-Specific Antigen of Ehrlichia risticii

Ehrlichia risticii, the causative agent of Potomac horse fever, has recently been isolated from many vaccinated horses with typical clinical signs of the disease. The heterogeneity of the E. risticii isolates obtained from the vaccinated horses necessitates the identification of the molecular basis of strain variations to elucidate the vaccine failure and to aid in the development of an efficient vaccine against this disease. As an attempt, two major cross-reacting surface antigen genes of 50- and 85-kDa antigens, present separately in strains 25-D (isolated in 1984) and 90-12 (isolated in 1990 from a vaccinated horse), respectively, were cloned and sequenced. A comparative sequence analysis revealed differences and similarities between these two antigens with strain-specific sizes (SSA). The 2.5- and 1.6-kb genes coding for the 85- and 50-kDa proteins, respectively, contained many different tandem repeats. The identical repeat motifs were more frequent in the middle of both genes, but the numbers and positions of the repeats were altogether different in the genes. Many of these direct repeats of both genes had exact sequence homology and coded for the same amino acids. The homology of the 5′- and 3′-flanking regions of the two genes was greater than that of the regions in the central part of the genes. A comparative analysis of the deduced amino acid sequences of these two antigen genes indicated eight common domains, which were designated identical domains. Although the sequence homologies of these identical domains were the same, the positions of the domains in their respective strains were completely different. This finding might be one of the bases of antigenic variation between the strains. In addition, there were a few unique regions in both antigen genes where no sequence homology existed. These specific regions were designated unique domains. The 50-kDa protein had two such unique domains, and the 85-kDa protein had six such unique domains. The presence of such unique domains contributed to the large size variation of these SSA. The cross-reactivity of recombinant proteins confirmed the presence of conserved epitopes between these two antigens. The SSA have been determined to be apparent protective antigens of E. risticii.     

Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.     

Association of Deficiency in Antibody Response to Vaccine and Heterogeneity of Ehrlichia risticii Strains with Potomac Horse Fever Vaccine Failure in Horses

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), which continues to be an important disease of horses. Commercial inactivated whole-cell vaccines are regularly used for immunization of horses against the disease. However, PHF is occurring in large numbers of horses in spite of vaccination. In a limited study, 43 confirmed cases of PHF occurred between the 1994 and 1996 seasons; of these, 38 (89%) were in horses that had been vaccinated for the respective season, thereby clearly indicating vaccine failure. A field study of horses vaccinated with two PHF vaccines indicated a poor antibody response, as determined by immunofluorescence assay (IFA) titers. In a majority of horses, the final antibody titer ranged between 40 and 1,280, in spite of repeated vaccinations. None of the vaccinated horses developed in vitro neutralizing antibody in their sera. Similarly, one horse experimentally vaccinated three times with one of the vaccines showed a poor antibody response, with final IFA titers between 80 and 160. The horse did not develop in vitro neutralizing antibody or antibody against the 50/85-kDa strain-specific antigen (SSA), which is the protective antigen of the original strain, 25-D, and the variant strain of our laboratory, strain 90-12. Upon challenge infection with the 90-12 strain, the horse showed clinical signs of the disease. The horse developed neutralizing antibody and antibody to the 50/85-kDa SSA following the infection. Studies of the new E. risticii isolates from the field cases indicated that they were heterogeneous among themselves and showed differences from the 25-D and 90-12 strains as determined by IFA reactivity pattern, DNA amplification finger printing profile, and in vitro neutralization activity. Most importantly, the molecular sizes of the SSA of these isolates varied, ranging from 48 to 85 kDa. These studies suggest that the deficiency in the antibody response to the PHF vaccines and the heterogeneity of E. risticii isolates may be associated with the vaccine failure.     

Cytopathogenic action ofEscherichia coli cells containing heterologous human type O(H) antigen on human cells in culture

The character of interaction between two enteropathogenic strains ofEscherichia coli of serotype 055K59 with human HeLa cells containing O(H) isoantigen was studied. On the addition of strainE. coli No. 5789, containing heterologous type O(H) antigen to a culture of HeLa cells, a cytopathogenic action was discovered on the third day of interaction in the presence of doses of bacterial cells of 2·10¹⁰, 2·10⁵, and 2·10⁴. A dose of 2·10³ bacterial cells ofE. coli did not give this effect. Strain No. 3827, not containing heterologous antigen of ABO type, had no cytopathogenic action in maximal, average, and small doses of bacterial cells. It is suggested that the cytopathogenic action of strain No. 5789 is connected with the presence of an antigen in this strain which is identical with the group antigen of the human cell culture studied.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 7, pp. 70–72, July, 1977.     

Detection of the phosphorylcholine epitope in streptococci,Haemophilusand pathogenicNeisseriaeby immunoblotting

The phosphorylcholine (PC) determinant inStreptococcus pneumoniaeis known to be linked to the cell wall polysaccharides (C-Ps) and to the lipoteichoic acid (LTA) (Forssman antigen) of the plasma membrane. Western blotting with two PC specific murine monoclonal antibodies (MAbs) designated 145,F-2 (IgM) and 147,A-1 (IgA) showed a similar ladder-like pattern for all examined strains ofS. pneumoniaeandStreptococcus mitis. Purified antigens run in parallel indicated that this ladder pattern is due to the PC of LTA. Unlike other techniques, Western blotting thus enables the identification of only one of the streptococcal structures carrying the PC epitope. Gram-negative organisms were also examined, and six of 11Haemophilus influenzaestrains reacted with the MAbs. For this species, unlike the streptococci, only one fast moving band was detected. Analyses by thin-layer chromatography (TLC) detected the PC epitope in lipopolysaccharide (LPS) fraction fromH. influenzae. Some strains of theNeisseriaceaefamily were also positive by Western blotting, but TLC and immunostaining did not detect the PC determinant in LPS.     

Immunocytochemical localization of the α-subunit of G-proteins in macrophages andEscherichia coli interacting with each other

The immunocytochemical localization of the α-subunit of G-proteins is established in murine macrophage-like P388D1 cells, inE. coli, and in L-forms ofE. coli. It is shown that the cytoplasmic concentration of G-proteins is increased in macrophages interacting with the bacteria. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3, pp. 309–311, March, 1996 Presented by S. V. Prozorovskii, Member of the Russian Academy of Medical Sciences     

Investigation of chloramphenicol acetyltransferase formation byEscherichia coli K-12 cells after changes in intracellular cyclic AMP concentration

The effect of cylic AMP, ACTH, and glucose on formation of the enzyme chloramphenicol acetyltransferase by cells ofEscherichia coli CSH-2/R222 andE. coli WZ-78/R222 (cya₈₅₅) was investigated. Glucose was shown to reduce synthesis of the enzyme inE. coli CSH-2/R222 by inducing catabolite repression; this could be overcome by 5 mM cyclic AMP and 1000 g/ml ACTH. Synthesis of the enzyme inE. coli WZ-78/R222 was resistant to catabolite repression and ACTH did not stimulate the synthesis of chloramphenicol acetyltransferase by this strain.Researh Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 294–295, March, 1976.     

Experession of human interleukin-10 inEscherichia coli cells

The human interleukin-10 gene has been synthesized by the chemical enzymatic method. Vectors for cytoplasmic and periplasmic expression of recombinant interleukin-10 have been obtained inE. coli cells. A high level of protein expression was found to be characteristic of only recombinant strains producing interleukin-10 as “fused” protein (fused with the N-terminal fragment of interleukin-3). Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 3, pp. 324–327, March, 1995 Presented by Yu. A. Romanov, Member of the Russian Academy of Medical Sciences). (Address for correspondence: Nauchnyi Pr. 8, 117246 Moscow, fax 331-01-01.     

The role of different protein components from the Haemophilus ducreyi cytolethal distending toxin in the generation of cell toxicity

Cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a multicomponent toxin, encoded by an operon consisting of three genes, cdtABC. To investigate the role of the individual products in generation of toxicity, recombinant plasmids were constructed allowing expression of each of the genes individually or in different combinations in Escherichia coli and Vibrio cholerae. Expression of all three genes (cdtABC) was necessary to generate toxicity on cells, and no activity was obtained using combinations in which only one or two of the genes were expressed. Of the individual gene products, the CdtA was shown to exist in two forms with an MW of 23 and 17 kDa, respectively. The CdtB protein alone resulted in DNase activity. CdtC purified from both toxic and non-toxic extracts (from strains expressingcdtCAB and cdtC, respectively) had a molecular weight of about 20 kDa and reacted with a CdtC-specific monoclonal antibody. However, the protein isoelectric point (pI) of CdtC from toxic preparations was about 1.5 pH units more basic than from non-toxic ones. Both forms were immunogenic giving rise to toxin-neutralizing antibodies. Toxicity was reconstructed by combining non-toxic cell sonicates from E. coli, expressing CdtA, CdtB and CdtC proteins individually. Only combinations including all three products gave toxicity, indicating that all are actively involved in the generation of toxic activity on cells. The reconstruction resulted in a 1.5 pH unit shift in the PI of CdtC, making it identical to that of the protein isolated from bacteria expressing cdtABC. The results showed that the CdtB component produces DNase activity, but cell toxicity depends on the involvement of the other two components of CDT and is associated with absorption of all three proteins by HEp-2 cells.     

Bacterial phenotypes mediated bymviAand their relationship to the mouse virulence ofSalmonella typhimurium

The focus of this study was the phenotypic characterization ofSalmonella typhimuriummutants lacking the function of the response regulatormviA. The inactivation ofmviA⁺(mviA: :kan) is shown to induce a significant change in the growth of most virulent strains, as reflected in the size of the colonies formed on agar plates. The colony phenotype observed in these strains has been designated as the small colony morphology (Scm⁺) phenotype. Mutants exhibiting the Scm⁺phenotype are shown to be significantly attenuated for virulence in susceptible (Ity^s) mice. The Scm⁺phenotype therefore provides anin vitrophenotypic marker formviA⁺activity. Further examination of Scm⁺mutants has revealed that they lack expression of a 55 kDa periplasmic protein which is detected in isogenicmviA⁺strains. This protein has been designatedmviA⁺relatedprotein A (MrpA) and was expressed in direct correlation with virulence in allS. typhimuriumstrains examined.     

Molecular cloning and characterization of a T24-like protein in Echinococcus multilocularis

One tetraspanin, designated as E24, was cloned from a full-length enriched vector-capping cDNA library of Echinococcus multilocularis metacestode. The amino acid sequence and phylogenetic analysis suggested that E24 is a T24-like protein. The crucial, functional large extracellular loop (LEL) domain of E24 was expressed and characterized using a polyclonal antiserum by Western blot and immunohistochemistry. The results showed that anti-recombinant-E24 (anti-recE24) antibody can specifically recognize approximately 25 kDa recombinant protein and 25 kDa cyst-extracted antigen; the germinal layer of both the protoscolex-free and protoscolex-formed cysts were intensely labeled by immunofluorescent antibody. This study revealed that E24 is an antigenic, germinal layer-located protein of E. multilocularis metacestode, implying for its potential in diagnostic and vaccine development.     

A western blot characterization of Mycobacterium bovis antigens recognized by cattle sera

The immune response to Mycobacterium bovis in cattle was assessed by Western blot. The antibody recognition pattern to M. bovis whole cell extracts and culture supernatant antigens was studied by using sera from M. bovis-infected (n=62) and healthy (n=38) cattle. Although the recognition patterns were highly variable, some proteins were regularly detected, mainly those with molecular masses of 17, 23, 28, 42, 66, 71 and 80 kDa in cellular extracts, and with molecular masses of 23 and 33 kDa in supernatants. Whole cell extract antigens were more frequently recognized than culture supernatant antigens. Healthy controls produced only a week antibody response.The antibody response was variable, depending on tuberculosis stage. In early stages very few antibodies were detected. A response against the 66-kDa stress protein was mounted in intermediate tuberculosis and remained stable in more advanced disease. In late diseases, the preferentially recognized antigens were a 28-kDa cellular protein and supernatant antigens.The 28-kDa protein was studied in some detail. As determined by using monoclonal antibodies, the 28-kDa protein is different from superoxide dismutase. This protein aggregated in stored cell extracts and was not totally transferred to nitrocellulose.The principal conclusions of this work are: (i) whole cell extract proteins are more frequently recognized than the secreted proteins and (ii) a 28-kDa protein is a major antigen in late disease.     

Assessment of the toxic and protective effects of initiators and inhibitors of free radical reactions using a wild-type strain ofEscherichia coli and a strain deficient for superoxide dismutase

The toxic effects of H₂O₂, paraquat, and oxidized low density lipoproteins are more expressed on superoxide dismutase-deficientE. coli strains than on its wild-type strains, and the effect of tert-butyl-hydroperoxide is less dependent on the presence or absence of this enzyme in the bacterium, whereas that of bleomycin does not depend on it at all. The toxicity of the agents increases in the following series: H₂O₂<oxidized low density lipoproteins<tert-butyl-hydroperoxide <paraquat≪bleomycin. A culture ofE. coli strains AB 1157 and JI 132 may be used for assessing the toxic effect of prooxidants, and anE. coli JI 132 culture with oxidative stress induced by prooxidants as a test system for detecting the potential antioxidants and assessing the mechanism of the action. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o , pp. 74–79, January, 1996 Presented by A. I. Archakov, Member of the Russian Academy of Medical Sciences     

Isolation and characterization of a 70 kDa protein fromMycobacterium avium

Mycobacterium aviumcomplex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein ofM. avium(Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) ofM. leprae(Ml) andM. tuberculosis(Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in anin vitroattachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-αby MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-αby these cells.     

Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) ofHelicobacter pylori

The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis ofHelicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed inEscherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts ofHelicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2 % and a specificity of 96.6 % compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections withHelicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.