scholarly journals Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovisBCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

2000 ◽  
Vol 68 (3) ◽  
pp. 1040-1047 ◽  
Author(s):  
Philippe Lefèvre ◽  
Olivier Denis ◽  
Lucas De Wit ◽  
Audrey Tanghe ◽  
Paul Vandenbussche ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Genomic Library ◽  
Signal Sequence ◽  
Interleukin 2 ◽  
Dna Vaccination ◽  
Mouse Strains ◽  
Mycobacterial Antigen ◽  
Cell Fractionation ◽  
Dna Encoding ◽  
Gene Encoding

ABSTRACT Using spleen cells from mice vaccinated with liveMycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.

scholarly journals Vaccination of Guinea Pigs with DNA Encoding the Mycobacterial Antigen MPB83 Influences Pulmonary Pathology but Not Hematogenous Spread following Aerogenic Infection with Mycobacterium bovis

2002 ◽  
Vol 70 (4) ◽  
pp. 2159-2165 ◽  
Author(s):  
Mark A. Chambers ◽  
Ann Williams ◽  
Graham Hatch ◽  
Dolores Gavier-Widén ◽  
Graham Hall ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Guinea Pigs ◽  
Vaccination Strategy ◽  
Control Program ◽  
Dna Vaccination ◽  
Mycobacterial Antigen ◽  
Lung Pathology ◽  
Dna Encoding ◽  
Hematogenous Spread ◽  
Bcg Vaccination

ABSTRACT Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. bovis could be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovis BCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.

Exon-Intron Organization in Genes of Earthworm and Vertebrate Globins

Science ◽  
1988 ◽  
Vol 240 (4850) ◽  
pp. 334-336 ◽  
Author(s):  
Sissy M. Jhiang ◽  
James R. Garey ◽  
Austen F. Riggs
Keyword(s):  
Signal Sequence ◽  
Globin Gene ◽  
Lumbricus Terrestris ◽  
Structure Characteristic ◽  
Globin Genes ◽  
Dna Encoding ◽  
Gene Encoding ◽  
Splice Junctions ◽  
Exon Structure ◽  
Exact Positions

The structure of an invertebrate, intron-containing globin gene has been determined as part of a study of the evolution of hemoglobin. The gene encoding chain c of Lumbricus terrestris hemoglobin has the two-intron, three-exon structure characteristic of vertebrate globin genes, and the exact positions of the splice junctions are conserved. The two introns interrupting the coding sequence are longer than those of known hemoglobins but shorter than myoglobin introns. The gene encodes a secretory preglobin containing a 16-residue signal peptide, as expected for an extracellular hemoglobin. However, no intron separates the DNA encoding the signal sequence from that of the globin sequence. The 3′ untranslated region of the Lumbricus gene is much longer than those of the genes for other hemoglobins and is similar to those found for myoglobins.

scholarly journals Mapping of Murine Th1 Helper T-Cell Epitopes of Mycolyl Transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis

2003 ◽  
Vol 71 (1) ◽  
pp. 483-493 ◽  
Author(s):  
S. D'Souza ◽  
V. Rosseels ◽  
M. Romano ◽  
A. Tanghe ◽  
O. Denis ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Interleukin 2 ◽  
T Cell Response ◽  
Mouse Strains ◽  
T Cell Epitopes ◽  
Dna Encoding ◽  
Amino Acid Region ◽  
Ifn Γ ◽  
Carboxy Terminal

ABSTRACT BALB/c (H-2d ) and C57BL/6 (H-2b ) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-γ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

scholarly journals Phage Shock Protein PspA of Escherichia coli Relieves Saturation of Protein Export via the Tat Pathway

2004 ◽  
Vol 186 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Matthew P. DeLisa ◽  
Philip Lee ◽  
Tracy Palmer ◽  
George Georgiou
Keyword(s):  
Genomic Library ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Protein A ◽  
Homologous Proteins ◽  
Gene Encoding ◽  
Protein Precursor ◽  
Twin Arginine Translocation ◽  
Tat Pathway

ABSTRACT Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.

scholarly journals Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus

2000 ◽  
Vol 23 (3) ◽  
pp. 685-688 ◽  
Author(s):  
P.W. Inglis ◽  
J.F. Peberdy ◽  
R.E. Sockett
Keyword(s):  
Agaricus Bisporus ◽  
Genomic Library ◽  
Signal Sequence ◽  
Selective Medium ◽  
Peptide Sequence ◽  
Accession Number ◽  
Gene Encoding ◽  
Expression Screening ◽  
Ewingella Americana ◽  
Cultivated Mushroom

We have isolated a gene encoding a chitinase (EC 3.2.1.14) from Ewingella americana, a recently described pathogen of the mushroom Agaricus bisporus. This gene, designated chiA (EMBL/Genbank/DDBJ accession number X90562), was cloned by expression screening of a plasmid-based E. americana HindIII genomic library in Escherichia coli using remazol brilliant violet-stained carboxymethylated chitin incorporated into selective medium. The chiA gene has a 918-bp ORF, terminated by a TAA codon, with a calculated polypeptide size of 33.2 kDa, likely corresponding to a previously purified and characterised 33-kDa endochitinase from E. americana. The deduced amino acid sequence shares 33% identity with chitinase II from Aeromonas sp. No. 10S-24 and 7.8% identity with a chitinase from Saccharopolyspora erythraeus. Homology to other chitinase sequences was otherwise low. The peptide sequence deduced from chiA lacks a typical N-terminal signal sequence and also lacks the chitin binding and type III fibronectin homology units common to many bacterial chitinases. The possibility that this chitinase is not primarily adapted for the environmental mineralisation of pre-formed chitin, but rather for the breakdown of nascent chitin, is discussed in the context of mushroom disease.

An endoglucanase from the anaerobic fungusOrpinomyces joyonii: characterization of the gene and its product

1997 ◽  
Vol 43 (5) ◽  
pp. 477-485 ◽  
Author(s):  
Jin-Hao Liu ◽  
L. Brent Selinger ◽  
You-Ji Hu ◽  
Kuo-Joan Cheng ◽  
Karen A. Beauchemin ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Genomic Library ◽  
Signal Sequence ◽  
Recombinant Protein Expression ◽  
Gene Encoding ◽  
Ph Optimum ◽  
Intronless Gene ◽  
Endoglucanase Gene ◽  
Domain Of Unknown Function ◽  
Bacteria And Fungi

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal fungus Orpinomyces joyonii. DNA sequence analysis of celA revealed an intronless gene encoding a typical signal sequence, an N-terminal catalytic domain, two repeated regions linked by a short Ser/Thr-rich linker and a domain of unknown function. The deduced amino acid sequence of the catalytic domain showed homology with the family 5 cellulases. While the catalytic domain of CelA was not homologous to the catalytic domain of the endoglucanase gene (EG3) from the ruminal bacterium Fibrobacter succinogenes, the repeated regions of CelA were very similar to the noncatalytic domain of EG3. This suggests that evolutionary shuffling of endoglucanase domains might occur among bacteria and fungi within the anaerobic ecosystem of the rumen. The celA gene was expressed in Escherichia coli, and the periplasmic endoglucanase was used for the characterization studies of the enzyme. CelA exhibited both endoglucanase and xylanase activities. Its pH optimum was 4 and the temperature optimum was 40 °C. Deletion analysis showed that the repeated sequences and C-terminal domain of CelA were not required for enzyme activity.Key words: endoglucanase, Orpinomyces joyonii, recombinant protein, expression.

scholarly journals Vaccination with DNA Encoding the Immunodominant LACK Parasite Antigen Confers Protective Immunity to Mice Infected with Leishmania major

1997 ◽  
Vol 186 (7) ◽  
pp. 1137-1147 ◽  
Author(s):  
Sanjay Gurunathan ◽  
David L. Sacks ◽  
Daniel R. Brown ◽  
Steven L. Reiner ◽  
Hughes Charest ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Protective Immunity ◽  
Leishmania Major ◽  
Protective Efficacy ◽  
Dna Vaccination ◽  
Attractive Alternative ◽  
Dna Immunization ◽  
Dna Encoding ◽  
Ifn Γ

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-γ (IFN-γ) production. Moreover, both the enhancement of IFN-γ production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

scholarly journals Investigation of Protein Export in Bifidobacterium breve UCC2003

2003 ◽  
Vol 69 (12) ◽  
pp. 6994-7001 ◽  
Author(s):  
Laura E. MacConaill ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
Fusion Proteins ◽  
Genomic Library ◽  
Shuttle Vector ◽  
Staphylococcal Nuclease ◽  
Cell Fractionation ◽  
Reporter System ◽  
Bifidobacterium Breve ◽  
Positive Effects ◽  
Encoding Gene ◽  
Export Signal

ABSTRACT The molecular interactions between the bifidobacterial cell and its natural environment, namely, the gastrointestinal tract of its host, are particularly important in understanding the presumed positive effects of Bifidobacterium on the health status of the host. In this study an export-specific reporter system, designed for use in gram-positive organisms and based on the use of the staphylococcal nuclease (Nuc) as a reporter, was employed to identify exported proteins in Bifidobacterium breve UCC2003. A B. breve genomic library of translational fusions to the Nuc-encoding gene devoid of its own export signal was established in the shuttle vector pFUN (I. Poquet, S. D. Ehrlich, and A. Gruss, J. Bacteriol. 180:1904-1912, 1998) and screened for bifidobacterial export signals. Sequence analysis of the fusion proteins obtained that displayed a nuclease-producing phenotype in both Lactococcus lactis and B. breve predicted the presence of a classical signal peptide and/or single or multiple transmembrane domains, thus indicating that some of the export signals in B. breve are comparable to those used in L. lactis. Cell fractionation studies, zymograms, nuclease assays, and Western blotting were employed to confirm the function of the predicted signals and to determine the location and activity of the exported fusion proteins in B. breve and/or L. lactis.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

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