European Commission COST/STD Initiative. Report of the expert panel IX. Vaccines against tuberculosis.
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Animals↗
Biomedical subjects
B Gicquel
Publications and source records attributed to B Gicquel.
The Mycobacterium tuberculosis purine biosynthetic pathway: isolation and characterization of the purC and purL genes.
Genes from the Mycobacterium tuberculosis purine biosynthetic pathway were identified using purine auxotrophic mutants of Mycobacterium smegmatis obtained by Tn611 transposon mutagenesis. Two approaches were followed in parallel. The first consisted of the complementation of the M. smegmatis purine auxotrophs using a M. tuberculosis H37Rv shuttle cosmid library. In the second approach, specific probes corresponding to the regions adjacent to the insertion sites of Tn611 in the M. smegmatis genome were used to screen a M. tuberculosis plasmid library by colony hybridization for inserts carrying homologous DNA fragments. Nucleotide sequence analysis of two M. tuberculosis genes isolated by these methods revealed high similarities with purC and purL genes from other bacterial and fungal sources. Transcriptional start sites were mapped for both genes, which revealed similar-10 boxes but with a higher GC content than the Escherichia coli sigma 70 consensus.
Amino Acid Sequence↗
Mycobacterium xenopi IS1395, a novel insertion sequence expanding the IS256 family.
An insertion sequence (IS) of Mycobacterium xenopi has been isolated and sequenced. This 1323 bp element, designated IS1395, is present in up to 18 copies in the M. xenopi genome and may be harboured in an M. xenopi extrachromosomal element. It encodes a putative transposase of 415 amino acids which displays sequence homology to the Staphylococcus aureus IS256 family. Members of this class of elements have been described in the genus Mycobacterium-for example IS1081 is present in the M. tuberculosis complex, IS1245-IS1311 in M. avium, and IS6120 in M. smegmatis; these elements exhibit an 89%, 45% and 16% amino acid identity with IS1395, respectively. Investigation of the host range of IS1395 by Southern blot analysis revealed additional IS1395-related repeated sequences in M. gordonae and M. celatum. Moreover, IS1395 represents a useful epidemiological tool for M. xenopi strain typing as it provides a diversity of restriction fragment length polymorphism patterns.
Amino Acid Sequence↗
Physical mapping of Mycobacterium bovis BCG pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M. bovis.
A Dral restriction map of the approximately 4.35 Mb circular chromosome of the vaccine strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 Dral fragments, ranging in size from 6 to 820 kb, using specific clones that spanned the Dral recognition sites as hybridization probes. The positions of 20 known genes were also established. Comparison of the resultant genome map with that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv revealed extensive global conservation of the genomes of these two members of the M. tuberculosis complex. Possible sites of evolutionary rearrangements were localized on the chromosome of M. bovis BCG Pasteur by comparing the Asnl restriction profile with that of M. tuberculosis H37Rv. When selected cosmids from the corresponding areas of the genome of M. tuberculosis H37Rv were used as hybridization probes to examine different BCG strains, wild-type M. bovis and M. tuberculosis H37Rv, a number of deletions up to 10 kb in size, insertions and other polymorphisms were detected. In addition to the known deletions covering the genes for the protein antigens ESAT-6 and mpt64, other genetic loci exhibiting polymorphisms or rearrangements were detected in M. bovis BCG Pasteur.
BCG Vaccine↗
Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.
The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.
Alleles↗
Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.
A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.
Acetyltransferases↗
Urease activity does not contribute dramatically to persistence of Mycobacterium bovis bacillus Calmette-Guérin.
Multiplication of BCGure-, an isogenic urease-negative mutant of Mycobacterium bovis BCG constructed by allelic exchange (J. M. Reyrat, F. X. Berthet, and B. Gicquel, Proc. Natl. Acad. Sci. USA 92:8768-8772, 1995), was examined in human macrophages and mice. Although ureolytic activity was not essential to BCGure-growth, a slight decrease in the multiplication and persistence of the mutated strain compared with wild-type BCG was observed in lungs of infected mice.
Animals↗
Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria.
Expression in mycobacteria of the structural gene sacB, which encodes the Bacillus subtilis levansucrase, was investigated. sacB expression is lethal to Mycobacterium smegmatis and Mycobacterium bovis BCG in the presence of 10% sucrose. sacB could thus be used as a counterselectable marker in mycobacteria.
Bacillus subtilis↗
Evaluation of tuberculosis transmission in a community by 1 year of systematic typing of Mycobacterium tuberculosis clinical isolates.
Interhuman transmission of Mycobacterium tuberculosis was investigated by using molecular typing, including restriction fragment length polymorphism with probes IS6110, DR (direct repeat) and PGRS (polymorphic GC-rich sequence) and a PCR method using the inverted repeat sequences of IS6110 as primers. From 105 patients hospitalized for tuberculosis during a 1-year survey in three hospitals in Paris, France, 111 isolates were collected and analyzed. Eighty-eight patients were infected with genetically different isolates, demonstrating the clonal heterogeneity of M. tuberculosis in these patients originating from various geographical areas. Fourteen patients were infected by strains clustered with identical fingerprints. An epidemiological relatedness was demonstrated for isolates from only seven of these patients. Thus, the typing of isolates from all tuberculous patients in hospitals during 1 year allows the detection of transmission in the general community. This would improve the case findings, thereby further improving the detection of outbreaks.
Adult↗
The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.
The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.
Alleles↗
Use of conjugative and thermosensitive cloning vectors for transposon delivery to Mycobacterium smegmatis.
Conjugative, thermosensitive shuttle plasmids capable of transfer from Escherichia coli to Mycobacterium smegmatis were constructed. They contain both an E. coli replicon and a thermosensitive derivative of the pAL5000 mycobacterial replicon. Using a temperature shift protocol, the conjugative plasmid, pJAZ11 was used to deliver the transposon Tn611 from E. coli into the chromosome of M. smegmatis. Analysis of transconjugants revealed the random insertion of the transposon.
Cloning, Molecular↗
BCG as a vector for the construction of multivalent recombinant vaccines.
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Animals↗
Recombinant BCG strains expressing the SIVmac251nef gene induce proliferative and CTL responses against nef synthetic peptides in mice.
CTL responses are known to be important for the control of HIV and SIV infections. Such responses are targeted against various components of these viruses including regulatory proteins like Nef. The SIVmac251nef gene was cloned in Mycobacterium bovis BCG under the control of P(AN), a promoter from Mycobacterium paratuberculosis. Nef was expressed as a fused polypeptide with ORF2, an open reading frame adjacent to P(AN). Mice inoculated with rBCG(SIVmac251nef) exhibited proliferative and CD8+ cytotoxic T-cell (CTL) responses against several Nef synthetic peptides. A mapping of the epitopes recognized by CTLs revealed that the central region of Nef is mainly involved in responses. This region had already been demonstrated to induce CTLs in experimentally SIV-infected macaques as well as in HIV-infected individuals. These results demonstrate the feasibility of constructing BCG vaccine strains expressing nef for eliciting cytotoxic responses.
Animals↗
Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice.
We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum beta-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.
Animals↗
Characterization of the Mycobacterium tuberculosis erp gene encoding a potential cell surface protein with repetitive structures.
Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. In this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar to that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. In addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pathogenic mycobacteria.
Amino Acid Sequence↗
Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.
The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.
Alkaline Phosphatase↗
Chromosomal DNA fingerprinting analysis using the insertion sequence IS6110 and the repetitive element DR as strain-specific markers for epidemiological study of tuberculosis in French Polynesia.
The polymorphism of Mycobacterium tuberculosis strains was evaluated in French Polynesia, an area with a low incidence of tuberculosis and a population which has been geographically stable during recent decades. Nonrepetitive strains isolated from 64 patients during 1991 and 1992 were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the insertion sequence IS6110 and the repetitive element DR as probes. Thirty-eight different IS6110 RFLP types were identified. They could be clustered in 11 groups. All the members of each group are identical or differ by one to three bands. All the other strains are gathered in the miscellaneous group. In some cases, transmission of strains with identical RFLP types between patients of the same family or between patients living in the same area was identified. Strains exhibiting similar IS6110 RFLP types also exhibited identical DR RFLP patterns, confirming that strains with similar types were genetically linked. Strains belonging to two different IS6110 clusters exhibited the same DR RFLP type. These data may also indicate a common origin for these strains and evolution to new IS6110 types. The results obtained in this study suggest that not only reactivation of latent tuberculous infections but also active transmissions are still occurring in French Polynesia.
Adolescent↗
[Genetic polymorphism of M. tuberculosis strains in Antananaviro].
The genetic polymorphism of the mycobacteria of the tuberculosis complex in the city of Antananarivo was studied on 126 strains isolated from positive microscopy pulmonary tuberculosis patients. The genetic profiles established using the RFLP technic and the IS6110 marker yielded 83 clusters of 1 to 29 strains. There were 34 strains with a IS6110 unique band profile of which 29 had a band located at 1.4-1.5 kb. These strains could be differentiated using a second marker, the DR marker. 3 strains with an unique IS6110 band located at 1.8-1.9 kb were identified as M. bovis. In general, there was no evident epidemiological relationship between the patients presenting with identical profiles. In the prison of Antananarivo, the IS6110 typing of 36 strains yielded 28 clusters of 1 to 3 strains. Excepting 2 clusters showing an internal contamination, the absence of profiles specific to the jail suggests that the patients were probably contaminated before their entrance. This preliminary study shows that the RFLP profiles of M. tuberculosis, using the IS6110 and the DR markers, were polymorphic enough for using this method to study the transmission in Antananarivo.
Cluster Analysis↗