Biomedical subjects

C Locht

Publications and source records attributed to C Locht.

At least 19 recordsLinked to original sources

PubMed2006

Modulation of the infant immune responses by the first pertussis vaccine administrations.

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis-tetanus-diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-gamma in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

Adhesins, Bacterial↗

PubMed2005

Recombinant Mycobacterium bovis BCG producing IL-18 reduces IL-5 production and bronchoalveolar eosinophilia induced by an allergic reaction.

BACKGROUND: Allergic reactions occur through the exacerbated induction of a Th2 cell type expression profile and can be prevented by agents favoring a Th1 profile. Bacillus Calmette-Guérin (BCG) is able to induce high IFN-gamma levels and has been shown to decrease experimentally induced allergy. The induction of IFN-gamma is mediated by interleukin (IL)-12 known to be secreted upon mycobacterial infections and can be enhanced by IL-18 acting in synergy with IL-12. OBJECTIVE: We evaluated the ability of a recombinant BCG strain producing IL-18 (rBCG) to modify the Th2 type responses in a murine model of ovalbumin (OVA)-dependent allergic reaction. METHODS: Mice were injected intraperitoneally or intranasally with OVA at days 0 and 15 and exposed to an OVA aerosol challenge at days 29, 30, 31 and 34. At days 0 and 15, two additional groups of mice received OVA together with 5 x 10(6) colony forming units of either rBCG or nonrecombinant BCG. RESULTS: A time-course analysis of OVA-specific immunoglobulin (Ig)E, IgG1 and IgG2a levels indicated no significant difference between the three groups of mice. However, following in vitro stimulation with OVA, lymph node cells from rBCG-treated mice produced less IL-5 and more IFN-gamma than those of mice injected with nonrecombinant BCG. In addition, 48 h after the last OVA challenge, a strong reduction of bronchoalveolar eosinophilia was found in the rBCG-injected mice compared to the nontreated or nonrecombinant BCG-treated groups. CONCLUSION: These results indicate that the production of IL-18 by rBCG may enhance the immunomodulatory properties of BCG that suppress pulmonary Th2 responses and, in particular, decrease airway eosinophilia.

Animals↗

PubMed2004

Clonal expansion of a globally disseminated lineage of Mycobacterium tuberculosis with low IS6110 copy numbers.

Knowledge of the clonal expansion of Mycobacterium tuberculosis and accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170 M. tuberculosis isolates with </=6 IS6110 insertions identified in Cape Town, South Africa, were characterized by principal genetic grouping, restriction fragment length polymorphism analysis, spoligotyping, IS6110 insertion site mapping, and variable-number tandem repeat (VNTR) typing. These analyses indicated that all but one of the isolates analyzed were members of principal genetic group 2 and of the same low-IS6110-copy-number lineage. The remaining isolate was a member of principal genetic group 1 and a different low-IS6110-copy-number lineage. Phylogenetic reconstruction suggests clonal expansion through sequential acquisition of additional IS6110 copies, expansion and contraction of VNTR sequences, and the deletion of specific direct-variable-repeat sequences. Furthermore, comparison of the genotypic data of 91 representative low-IS6110-copy-number isolates from Cape Town, other southern African regions, Europe, and the United States suggests that certain low-IS6110-copy-number strain spoligotypes and IS6110 fingerprints were acquired in the distant past. These clones have subsequently become widely disseminated and now play an important role in the global tuberculosis epidemic.

DNA Transposable Elements↗

PubMed2003

Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

Bordetella pertussis↗

PubMed2003

Adjuvant activity of free Bordetella pertussis filamentous haemagglutinin delivered by mucosal routes.

The development of safe and potent mucosal adjuvants remains a major objective in vaccinology. The potential usefulness of filamentous haemagglutinin (FHA) of Bordetella pertussis as an adjuvant was assessed in a mouse model. The glutathione-S-transferase of Schistosoma mansoni (Sm28GST) was used for intranasal administration, while the gut-resistant keyhole limpet haemocyanin (KLH) was administrated by the oral route. For both antigens, coadministration with FHA increased antigen-specific immunoglobulin titres. This adjuvant effect did not require chemical cross-linking or direct interaction between FHA and the antigen tested. FHA also behaved as an adjuvant by the subcutaneous route, indicating that its adjuvanticity is not restricted to binding to mucosal surfaces. The FHA-induced adjuvanticity was also observed in mice with high anti-FHA antibody titres as a result of antipertussis vaccination, indicating that pre-existing anti-FHA antibodies do not impair FHA adjuvanticity. No mRNA coding for proinflammatory cytokines was induced in the lungs after intranasal FHA administration. However, an increase in the levels of mRNAs coding for B7-1, transforming growth factor (TGF)-beta and major histocompatibility complex (MHC)-II was detected in the lungs after FHA administration. Although the molecular mechanisms of the FHA-induced adjuvanticity remain to be elucidated, the data presented here indicate that this adhesin, already assessed for human use as a pertussis vaccine constituent, represents a promising adjuvant to improve the humoral immune response when given by mucosal routes.

Adhesins, Bacterial↗

PubMed2001

Tetanus toxin fragment C-specific priming by intranasal infection with recombinant Bordetella pertussis.

As an alternative to parenteral administration, mucosal administration offers several advantages including the ease of administration, safety and the ability to induce mucosal immunity. As a first step towards nasal administration of important childhood vaccines, we have previously developed attenuated Bordetella pertussis strains able to protect mice against pertussis upon nasal vaccination. Since pertussis vaccines are generally combined with tetanus and diphtheria vaccines, we constructed recombinant B. pertussis strains producing the non-toxic protective tetanus toxin fragment C (TTFC). TTFC was genetically fused to the N-terminal domain of the B. pertussis filamentous haemagglutinin. The hybrid gene was introduced into B. pertussis both on a multi-copy replicative plasmid and as a single copy inserted into the chromosome of a pertussis toxin-producing strain and a toxin-deficient attenuated strain. The hybrid protein was secreted by the recombinant strains. However, the recombinant multi-copy plasmid was unstable in vivo, and immunisation could only be carried out with the strains containing the single-copy chromosomal integration. Both the toxin-producing and the toxin-deficient recombinant B. pertussis strains were able to prime mice for the production of anti-TTFC serum antibodies upon intranasal administration, suggesting the feasibility of using recombinant attenuated B. pertussis for the development of combined childhood vaccines.

Administration, Intranasal↗

PubMed2001

Nasal vaccination using live bacterial vectors.

Live recombinant bacteria represent an attractive means to induce both mucosal and systemic immune responses against heterologous antigens. Several models have now been developed and shown to be highly efficient following intranasal immunization. In this review, we describe the two main classes of live recombinant bacteria: generally recognized as safe bacteria and attenuated strains derived from pathogenic bacteria. Among the latter, we have differentiated the bacteria, which do not usually colonize the respiratory tract from those that are especially adapted to respiratory tissues. The strategies of expression of the heterologous antigens, the invasiveness and the immunogenicity of the recombinant bacteria are discussed.

Administration, Intranasal↗

PubMed2001

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway.

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.

Adhesins, Bacterial↗

PubMed2001

The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination.

Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.

Animals↗

PubMed2001

Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II.

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed2001

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology.

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Bacterial Typing Techniques↗

PubMed2001

Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection.

In vivo delivery of DNA encoding antigens is a simple tool to induce immune responses against pathogens. This approach to vaccination also offers the possibility to codeliver plasmids encoding immunomodulatory molecules in order to drive immune responses towards optimal protective effects. In the murine model of Schistosoma mansoni infection, vaccination inducing a Th1 profile has been shown to be protective. In this study, we used a plasmid encoding the Th1-promoting cytokine IL-18, since we observed that percutaneous infection of Balb/c mice strongly induced the production of IL-18 mRNA in the skin. Intradermal injection of the IL-18-encoding plasmid prior to infection did not interfere with parasite migration through the skin although it led to a local and transient cellular infiltration. When the IL-18-encoding plasmid was codelivered with a S. mansoni glutathione S-transferase (Sm28GST)-encoding plasmid, a 30-fold increase of antigen-specific IFN-gamma secretion by spleen cells was observed in comparison to spleen cells from mice that had received only the Sm28GST-encoding plasmid. This immunostimulatory effect was related to a significant protective effect (28% reduction in egg laying and 23% reduction in worm burden) which was attributed to a cooperative effect between both plasmids. Therefore, this study shows that codelivery of an IL-18-encoding plasmid with an antigen-encoding plasmid can stimulate specific cellular responses and induce protective effects against S. mansoni infection.

Animals↗

PubMed2001

Bordetella pertussis, molecular pathogenesis under multiple aspects.

Recent studies, including those based on genomics, have demonstrated that besides toxins and adhesins, Bordetella pertussis uses many additional virulence determinants. Most of them are part of the BvgAS regulon, although some, in particular iron-uptake systems, are independent of BvgAS. They are regulated by iron, although in one case, the production of a siderophore receptor could be linked to the BvgAS regulon.

Adhesins, Bacterial↗

PubMed2001

Characterization of the type III secretion locus of Bordetella pertussis.

Multiple sequence comparisons of proteins of the LcrD/FlbF family allowed the design of primers that specifically amplify sequences coding for type III secretion components. Amplification of Bordetella pertussis DNA with these primers yielded a fragment that was further used as a probe for screening a genomic library. The nucleotide sequence of a positive clone revealed a 2100-bp gene, called bcrD, which specifies a 75-kDa polypeptide homologous to the Yersinia LcrD protein. Chromosome walking allowed the characterization of a 35-kb DNA segment that contains the entire locus and flanking housekeeping genes. The B. pertussis type III secretion locus consists of more than 30 open reading frames (ORFs), most of which are identical to annotated genes of Bordetella spp and share similarities with known type III secretion genes of related bacteria. In order to assess the function of this locus, we engineered a bcrD null mutant. However, none of the tested phenotypes, such as protein secretion, cellular invasion, cytotoxicity or mouse lung colonization, differentiated the mutant from its parental strain. Studies of bcrD and bscN expressions indicated that, under our experimental conditions, these genes are not expressed in vitro. Restriction analyses on pulsed-field gel electrophoresis allowed the type III locus mapping at coordinate position 1,590 kb on the Tohama I strain chromosome.

Animals↗

PubMed2001

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

Antibodies, Bacterial↗

PubMed2001

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins.

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.

Amino Acid Sequence↗

PubMed2001

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins.

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.

Adhesins, Bacterial↗

PubMed2001

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin.

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis. The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-alpha production in bronchoalveolar lavage (BAL) fluids of B. pertussis-infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-alpha.

Animals↗