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B Gicquel

Publications and source records attributed to B Gicquel.

At least 55 records · Page 3Linked to original sources
PubMed1998

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

Cloning, Molecular↗
PubMed1998

Genetic advances for studying Mycobacterium tuberculosis pathogenicity.

Tuberculosis remains the greatest cause of death worldwide because of a single pathogen. Despite its importance, the genetic basis of the pathogenicity of Mycobacterium tuberculosis remains poorly understood, mainly because the most productive investigative approach, molecular genetic analysis, has been severely hampered by a lack of efficient tools. However, significant advances, including the development of methods for inactivating genes and studying their expression with reporter genes, have been recently made. This progress may lead to opportunities for developing new vaccines and antituberculous drugs. The aim of this review is to examine the present state of the art in mycobacterial molecular genetics and pinpoint some expected or promising areas for future research.

Animals↗
PubMed1998

Identification of genetic loci implicated in the survival of Mycobacterium smegmatis in human mononuclear phagocytes.

A luminescence-based procedure that permits the rapid evaluation of the survival of mycobacteria within mononuclear phagocytes was developed and used to screen insertional mutants of Mycobacterium smegmatis for their ability to survive in human monocyte-derived macrophages. Among the 5000 mutants tested, eight mutants were identified that demonstrated impaired intracellular survival in human macrophages but that grew normally in the absence of cells. For each mutant, a portion of the gene interrupted by the transposition event was amplified by ligand-mediated PCR and sequenced. In all cases, the existence of homologous genes of as yet unknown function were identified in the Mycobacterium tuberculosis genome. Complementation of the mutant mycobacterial strains with cosmids containing the homologous loci from M. tuberculosis restored normal intracellular growth in three of the four mutants tested, supporting the idea that these loci contain genes that are important for intracellular survival. This study demonstrates the feasibility of directly screening mutant mycobacterial strains to identify genes coding for activities necessary for the intracellular survival in human mononuclear phagocytes, an important initial step in the identification of potential targets for new therapeutic agents.

Base Sequence↗
PubMed1997

Recombinant Mycobacterium bovis BCG producing the N-terminal half of SIVmac251 Env antigen induces neutralizing antibodies and cytotoxic T lymphocyte responses in mice and guinea pigs.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) represents a good candidate for the development of vaccines against AIDS. Several HIV or SIV genes including nef, gag, and env have already been expressed by rBCG strains and shown to induce strong humoral and cellular immune responses in experimental animals. Because a broad immune response directed to multiple HIV/SIV antigens is highly desirable in order to develop effective vaccines, we have also investigated the immune response induced by an rBCG strain expressing a large N-terminal portion of the SIVmac251 Env gp110-encoding gene. The rBCG(SIVmac251Env) strain obtained was able to induce strong CTL responses in mice as well as humoral immune responses in mice and guinea pigs immunized by parenteral routes. The anti-gp110 IgGs produced were able to neutralize in vitro growth of virulent SIVmac251 field isolates. Moreover, guinea pigs immunized by the oral route produced significant levels of anti-gp110 IgAs in the feces, demonstrating that rBCG is able to induce local humoral immunity in the intestinal mucosa. These data provide further evidence of the utility of BCG as a candidate vaccine vector against AIDS.

Animals↗
PubMed1997

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Alleles↗
PubMed1997

Contribution of beta-lactamase production to the resistance of mycobacteria to beta-lactam antibiotics.

Mycobacterium fallax (M. fallax) is naturally sensitive to many beta-lactam antibiotics (MIC < 2 microg/ml) and devoid of beta-lactamase activity. In this paper, we show that the production of the beta-lactamase of Mycobacterium fortuitum by M. fallax significantly increased the MIC values for good substrates of the enzyme, whereas the potency of poor substrates or transient inactivators was not modified. The rates of diffusion of beta-lactams through the mycolic acid layer were low, but for all studied compounds the half-equilibration times were such that they would only marginally affect the MIC values in the absence of beta-lactamase production. These results emphasize the importance of enzymatic degradation as a major factor in the resistance of mycobacteria to penicillins.

Anti-Bacterial Agents↗
PubMed1997

Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2')-Ic gene from Mycobacterium tuberculosis and the aac(2')-Id gene from Mycobacterium smegmatis.

The genus Mycobacterium comprises clinically important pathogens such as M. tuberculosis, which has reemerged as a major cause of morbidity and mortality world-wide especially with the emergence of multidrug-resistant strains. The use of fast-growing species such as Mycobacterium smegmatis has allowed important advances to be made in the field of mycobacterial genetics and in the study of the mechanisms of resistance in mycobacteria. The isolation of an aminoglycoside-resistance gene from Mycobacterium fortuitum has recently been described. The aac(2')-Ib gene is chromosomally encoded and is present in all isolates of M. fortuitum. The presence of this gene in other mycobacterial species is studied here and genes homologous to that of M. fortuitum have been found in all mycobacterial species studied. In this report, the cloning of the aac(2')-Ic gene from M. tuberculosis H37Rv and the aac(2')-Id gene from M. smegmatis mc(2)155 is described. Southern blot hybridizations have shown that both genes are present in all strains of this species studied to date. In addition, the putative aac(2')-Ie gene has been located in a recent release of the Mycobacterium leprae genome. The expression of the aac(2')-Ic and aac(2')-Id genes has been studied in M. smegmatis and only aac(2')-Id is correlated with aminoglycoside resistance. In order to elucidate the role of the aminoglycoside 2'-N-acetyltransferase genes in mycobacteria and to determine whether they are silent resistance genes or whether they have a secondary role in mycobacterial metabolism, the aac(2')-Id gene from M. smegmatis has been disrupted in the chromosome of M. smegmatis mc(2)155. The disruptant shows an increase in aminoglycoside susceptibility along with a slight increase in the susceptibility to lysozyme.

Acetyltransferases↗
PubMed1997

Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients.

The phoA gene fusion methodology permitted the identification of a new Mycobacterium tuberculosis exported protein, Des. This protein has significant sequence similarities to plant acyl-acyl carrier protein desaturases, which are enzymes involved in general fatty acid biosynthesis as well as in mycolic acid biosynthesis in mycobacteria. As shown by Western blot and enzyme-linked immunosorbent assay experiments, the Des protein is a major B-cell antigen that was recognized by all the tuberculous M. tuberculosis- and M. bovis-infected human patients tested.

Amino Acid Sequence↗
PubMed1997

Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen.

Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various inbred strains of mice to recombinant BCG expressing beta-galactosidase. These experiments demonstrated that BALB/c mice developed strong antibody responses against BCG expressing beta-galactosidase under the control of two different promoters. In contrast, C57BL/6, C3H, and CBA mice produced high anti-beta-galactosidase antibody titers only when immunized with recombinant BCG expressing beta-galactosidase under the control of the pblaF* promoter, which induced the production of high levels of this antigen. This difference in mouse responsiveness to recombinant BCG was not due to innate resistance to BCG infection, since similar immune responses were induced in Ity(r) and Ity(s) congenic strains of mice. In contrast, the analysis of anti-beta-galactosidase antibody responses of H-2 congenic mice in two different genetic backgrounds demonstrated that H-2 genes are involved in the immune responsiveness to beta-galactosidase delivered by recombinant BCG. Together, these results demonstrate that immune responses to an antigen delivered by recombinant BCG are under complex genetic influences which could play a crucial role in the efficiency of future recombinant BCG vaccines.

Animals↗
PubMed1997

Rapid discrimination of Mycobacterium tuberculosis complex strains by ligation-mediated PCR fingerprint analysis.

A ligation-mediated PCR (LMPCR) method for the amplification of sequences flanking the IS6110 of the Mycobacterium tuberculosis complex has been developed. The method uses one primer specific for IS6110 and a second specific for a linker ligated to SalI-restricted genomic DNA. LMPCR is a rapid screening method, valuable for the fingerprinting of M. tuberculosis complex strains.

Bacterial Typing Techniques↗
PubMed1997

Evaluation of spoligotyping in a study of the transmission of Mycobacterium tuberculosis.

Spoligotyping (for spacer oligotyping) is an easy, economical, and rapid way of typing Mycobacterium tuberculosis complex strains with the DR spacer markers (J. Kamerbeek et al., J. Clin. Microbiol. 35:907-914, 1997; D. van Soolingen et al., 33:3234-3248, 1995). The stability of the markers was demonstrated by showing that all the Mycobacterium bovis BCG strains tested gave the same spoligotyping pattern. None of the 42 atypical mycobacterial strains tested gave a spoligotyping signal, indicating the specificity of the technique for M. tuberculosis complex. The utility of the spoligotyping method was demonstrated by analyzing 106 isolates of M. tuberculosis obtained over 1 year in three Paris hospitals. The results obtained by this technique were compared to those obtained by Torrea et al. (G. Torrea et al., J. Clin. Microbiol. 34:1043-1049, 1996) by IS6110-based restriction fragment length polymorphism (RFLP) analysis. Strains from patients with epidemiological relationships that were in the same IS6110-RFLP cluster were also in the same spoligotyping group. Spoligotyping was more discriminative than RFLP analysis for strains with one or two copies of IS6110. RFLP analysis did not discriminate between the nine strains with one or two IS6110 bands with no known epidemiological relation, whereas spoligotyping distinguished between eight different types. IS6I10-RFLP analysis split some of the spoligotyping clusters, particularly when the IS6110 copy number was high. Therefore, we propose a strategy for typing M. tuberculosis strains in which both markers are used.

Bacterial Typing Techniques↗
PubMed1997

Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice.

Recombinant live Mycobacterium bovis BCG vectors (rBCG) induce strong cellular and humoral immune responses against various antigens after either systemic or oral immunization of mice. Cytotoxic T-lymphocyte (CTL) responses may contribute to the control of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infections whose portal of entry is the gastrointestinal or genital mucosa. In this study, we immunized BALB/c mice with a recombinant BCG SIV nef and observed its behavior in oropharyngeal and target organ lymphoid tissues. The cellular immune responses, particularly the intestinal intraepithelial and systemic CTL responses, were investigated. The results showed that rBCG SIV nef translocated the oropharyngeal mucosa and intestinal epithelium. It diffused to and persisted in target lymphoid organs. Specific SIV Nef peptide proliferative responses and cytokine production were observed. Strong systemic and mucosal CTL responses were induced. In particular, we demonstrated direct specific anti-Nef CTL in intestinal intraepithelial CD8beta+ T cells. These findings provide evidence that orally administered rBCG SIV nef may contribute to local defenses against viral invasion. Therefore, rBCG SIV nef could be a candidate vaccine to protect against SIV infection and may be used to develop an oral rBCG HIV nef vaccine.

Administration, Oral↗
PubMed1997

No evidence for linkage between leprosy susceptibility and the human natural resistance-associated macrophage protein 1 (NRAMP1) gene in French Polynesia.

In order to determine whether a human homolog (NRAMP1) to a murine candidate gene for resistance to mycobacteria influences susceptibility to human disease, we analyzed data from seven multicase leprosy families (84 individuals) from French Polynesia for linkage markers within the NRAMP1 gene and leprosy per se. Individual family members were typed at nine polymorphic loci within NRAMP1. In addition, three physically linked, polymorphic microsatellite markers-D2S104, D2S173 and D2S1471-were also typed. Linkage analyses were done using affected sibpair and LOD score methods employing different modes of inheritance with full and reduced penetrance. The results of this study strongly suggest that NRAMP1 is not linked to leprosy susceptibility in the French Polynesian families tested.

Alleles↗
PubMed1996

Positive selection of allelic exchange mutants in Mycobacterium bovis BCG.

sacB expression is lethal to mycobacteria in the presence of sucrose. It can therefore serve as 1 counter-selectable marker for positive selection of gene replacement events as demonstrated in the fast-growing Mycobacterium smegmatis. With this methodology, a sucrose counter-selectable vector was used to deliver, into the Mycobacterium bovis BCG genome, an inactivated copy (ureC::Km) of the ureC gene encoding the mycobacterial urease. A two-step selection procedure on 2% sucrose allowed the positive selection of gene exchange mutants. This technique should thus be extremely useful for the genetic analysis of pathogenic mycobacteria.

Alleles↗