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C Locht

Publications and source records attributed to C Locht.

At least 37 records · Page 2Linked to original sources
PubMed2001

Role of ADP-ribosyltransferase activity of pertussis toxin in toxin-adhesin redundancy with filamentous hemagglutinin during Bordetella pertussis infection.

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two major virulence factors of Bordetella pertussis. FHA is the main adhesin, whereas PT is a toxin with an A-B structure, in which the A protomer expresses ADP-ribosyltransferase activity and the B moiety is responsible for binding to the target cells. Here, we show redundancy of FHA and PT during infection. Whereas PT-deficient and FHA-deficient mutants colonized the mouse respiratory tract nearly as efficiently as did the isogenic parent strain, a mutant deficient for both factors colonized substantially less well. This was not due to redundant functions of PT and FHA as adhesins, since in vitro studies of epithelial cells and macrophages indicated that FHA, but not PT, acts as an adhesin. An FHA-deficient B. pertussis strain producing enzymatically inactive PT colonized as poorly as did the FHA-deficient, PT-deficient strain, indicating that the ADP-ribosyltransferase activity of PT is required for redundancy with FHA. Only strains producing active PT induced a local transient release of tumor necrosis factor alpha (TNF-alpha), suggesting that the pharmacological effects of PT are the basis of the redundancy with FHA, through the release of TNF-alpha. This may lead to damage of the pulmonary epithelium, allowing the bacteria to colonize even in the absence of FHA.

Adhesins, Bacterial↗
PubMed2001

Production of Neisseria meningitidis transferrin-binding protein B by recombinant Bordetella pertussis.

Neisseria meningitidis serogroup B infections are among the major causes of fulminant septicemia and meningitis, especially severe in young children, and no broad vaccine is available yet. Because of poor immunogenicity of the serogroup B capsule, many efforts are now devoted to the identification of protective protein antigens. Among those are PorA and, more recently, transferrin-binding protein B (TbpB). In this study, TbpB of N. meningitidis was genetically fused to the N-terminal domain of the Bordetella pertussis filamentous hemagglutinin (FHA), and the fha-tbpB hybrid gene was expressed in B. pertussis either as a plasmid-borne gene or as a single copy inserted into the chromosome. The hybrid protein was efficiently secreted by the recombinant strains, despite its large size, and was recognized by both anti-FHA and anti-TbpB antibodies. A single intranasal administration of recombinant virulent or pertussis-toxin-deficient, attenuated B. pertussis to mice resulted in the production of antigen-specific systemic immunoglobulin G (IgG), as well as local IgG and IgA. The anti-TbpB serum antibodies were of the IgG1, IgG2a, and IgG2b isotypes and were found to express complement-mediated bactericidal activity against N. meningitidis. These observations indicate that recombinant B. pertussis may be a promising vector for the development of a mucosal vaccine against serogroup B meningococci.

Adhesins, Bacterial↗
PubMed2001

Expression of the putative siderophore receptor gene bfrZ is controlled by the extracytoplasmic-function sigma factor BupI in Bordetella bronchiseptica.

A new gene from Bordetella bronchiseptica, bfrZ encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay. A bfrZ null mutant was constructed by allelic exchange. The protein profile of this mutant is similar to that of the wild-type parent strain. The BfrZ(-)-BfrZ(+) isogenic pair was tested for utilization of 132 different siderophores as iron sources. None of these iron sources acted as a ligand for BfrZ. Translational bfrZ::phoA and transcriptional bfrZ::lacZ fusions were introduced into the B. bronchiseptica bfrZ locus. No alkaline phosphatase or beta-galactosidase activity was detected. Sequence analysis of the bfrZ upstream region revealed the presence of two tightly linked genes, bupI and bupR. Both of these genes are located downstream from a Fur-binding sequence. BupI is homologous to Escherichia coli FecI and Pseudomonas putida PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions. BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane. Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension. We found that bfrZ is not transcribed when bupI and bupR are expressed at the same level. However, overexpression of bupI from a multicopy plasmid triggers bfrZ transcription, and under these conditions BfrZ was detected in membrane fractions. By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that bfrZ expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope.

Alleles↗
PubMed2001

Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units.

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

Automation↗
PubMed2001

[Cellular and acellular anti-pertussis vaccines].

Since 1940, vaccination with whole cell vaccines, composed of Bordetella pertussis extracts, has dramatically reduced the incidence of whooping cough. However, the occurrence of side effects has stimulated research that has resulted in the design of new, acellular vaccines. These vaccines are composed of adhesins, such as filamentous haemagglutinin and pertactin, and detoxified pertussis toxin. Detoxification can be achieved by genetic engineering resulting in alterations of specific amino acid residues that are involved in the enzymatic activity or in the target-cell receptor binding activity of the toxin. Assessment on laboratory animals and in clinical trials have indicated that these acellular vaccines are almost as protective as the whole cell vaccines, and that they are much less reactogenic. However, these vaccines are more expensive and still need multiple injections to achieve optimal protection. An interesting alternative would be the construction of attenuated strains able to protect after a single intranasal administration. In addition, the construction of recombinant attenuated strains would perhaps simultaneously protect against whooping cough and other infectious or parasitic diseases.

Animals↗
PubMed2000

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.

Adhesins, Bacterial↗
PubMed2000

Activation of the pro-drug ethionamide is regulated in mycobacteria.

The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.

Amino Acid Sequence↗
PubMed2000

Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine.

Many different HIV-1 vaccine strategies have been developed, but as yet none has been completely successful. Promising results from combined DNA prime/protein boost vaccines have been reported. Specific immune responses generated by DNA vaccines can be modulated by the co-delivery of genes coding for cytokines. In this study, we have used the intradermal route by needle injection of a plasmid coding for the HIV-1 Nef accessory protein. We show that DNA prime/protein boost vaccine combinations increase the humoral and cellular immune responses against HIV-1 Nef and that the co-injection of DNA encoding Interleukin-18 (IL-18) modulates the specific immune response towards a Th1 type.

AIDS Vaccines↗
PubMed2000

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin.

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.

Base Sequence↗
PubMed2000

Genomics of Bordetella pertussis toxins.

Bordetella pertussis, the etiologic agent of whooping cough, produces numerous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT). PTX is composed of five different subunits organised in a typical A-B type structure of which the A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity. The secretion of this toxin requires nine other genes (ptl) organised in an operon together with the five structural genes of PTX. To further characterise the genetic locus of this major virulence factor, we analysed the ptx/ptl upstream and downstream sequences. Comparison of these regions between three species of Bordetella (B. pertussis, Bordetella parapertussis and Bordetella bronchiseptica) revealed differences in the upstream region. Analysis of two strains of B. bronchiseptica naturally lacking the ptx genes showed that only the ptx/ptl genes were deleted in these strains, and that the upstream and downstream regions were conserved. Upstream of the PTX structural genes and the promoter, an open reading frame (bugT) was identified, the product of which is homologous with putative proteins from several other Gram-negative organisms. Detailed analysis of the genome of B. pertussis which is currently sequenced at the Sanger Centre revealed the presence of 90 genes coding for proteins homologous to BugT, which qualifies the bug gene family as the most populated one of Bordetella. These bug genes are located in various genetic environments, including the proximities of genes coding for other toxins, such as DNT and AC. The Bug proteins are highly conserved in terms of size and periodicity of predicted secondary structure elements, but have also a high variability in their amino acid composition reflected in their wide range of isoelectric points. The function of these genes which is currently unknown is under investigation. To characterise the expression and regulation of these genes, as well as of novel putative B. pertussis virulence factors, we designed a transcriptional fusion vector to be inserted in precise locations of the B. pertussis chromosome by homologous recombination. The reporter gene present in this vector allowed us to show that at least some of the bug genes are expressed.

Adenylate Cyclase Toxin↗
PubMed2000

Intracellular trafficking and membrane translocation of pertussis toxin into host cells.

The translocation of the pertussis toxin (PTX) S1 subunit into the cytoplasm of host cells was analysed in CHO cells producing S1 fused to a signal peptide. This protein channelled into the endoplasmic reticulum (ER) by the signal peptide, was found to ADP-ribosylate its target G proteins, suggesting that membrane translocation can occur from the ER and does not require the B oligomer. Similar results were obtained with a C-terminally truncated S1 subunit, indicating that this hydrophobic tail is not involved in the translocation mechanism. We also analysed the activity of two PTX mutants in which the S3 and S2 subunits were substituted for each other. The mutant protein containing two S3 subunits (PTXAS2) presented a decreased binding to fetuin or haptoglobin but higher in vivo activity than the wild-type PTX, suggesting that replacement of S2 by S3 favours the targeting of PTX to the compartment where translocation occurs and/or the dissociation of S1 from the B oligomer, thereby leading to a better translocation of S1 into the cytoplasm.

Adenosine Diphosphate Ribose↗
PubMed2000

Live bacterial vectors for intranasal delivery of protective antigens.

To exploit the advantages of mucosal, particularly intranasal, vaccination, several live bacterial vectors have been developed and shown to elicit strong immune responses, including protective immunity against viruses, bacteria or parasites. Two main categories can be distinguished; those that are based on commensal bacteria, such as lactococci, lactobacilli or certain streptococci and staphylococci, and those that are based on attenuated pathogens, such as Salmonella, BCG and Bordetella. The quality of the immune responses may vary between the vector systems, but in most cases the immune responses obtained after intranasal administration are stronger than those obtained after oral administration of the same vaccines.

Journal Article↗
PubMed2000

Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome.

Mycobacterial interspersed repetitive units (MIRUs) are 40-100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite-like structures evolve slowly in mycobacterial populations.

Base Sequence↗
PubMed2000

Genital antibody responses in mice after intranasal infection with an attenuated candidate vector strain of Bordetella pertussis.

Intranasal administration of live attenuated Bordetella pertussis, from which the pertussis toxin gene has been deleted, has previously been shown to give rise to high levels of serum immunoglobulin G (IgG) antibodies against both the protective antigen filamentous hemagglutinin (FHA) and heterologous antigens genetically fused to FHA. Here, we extend these results by demonstrating that anti-FHA IgA and IgG antibodies are also produced in the genital tract of mice, both in the vagina and in the uterus, after a single intranasal administration of B. pertussis. By comparing the immune responses induced after infection with wild-type virulent B. pertussis with that induced by infection with an attenuated pertussis toxin-deficient strain, we conclude that pertussis toxin produced by the virulent bacteria does not modify antibody production to FHA in the genital tract of B. pertussis-infected mice. The intranasal infection with either the attenuated or the virulent B. pertussis strain also led to the development of immunologic memory that could be efficiently boosted with purified FHA administered either intranasally or intravaginally to give rise to a significant increase in the levels of specific IgA and IgG produced locally in the genital tract, as well as of specific antibodies in the serum. These observations suggest that attenuated B. pertussis could be a promising vector for intranasal administration to induce antibody responses against antigens from sexually transmitted pathogens fused to FHA.

Adhesins, Bacterial↗
PubMed2000

Bordetella pertussis TonB, a Bvg-independent virulence determinant.

In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm. Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment. The tight organization of the three genes suggests that they are cotranscribed. A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions. Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively. A B. pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized. The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources. Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair. The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent. Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain.

Amino Acid Sequence↗
PubMed2000

Ineffective cellular immune response associated with T-cell apoptosis in susceptible Mycobacterium bovis BCG-infected mice.

It has previously been reported that inhibition of delayed-type hypersensitivity-mediating functions of T cells during mycobacterial infection in mice is haplotype dependent. In the present study, we show that Mycobacterium bovis BCG infection induced, in susceptible C57BL/6 and BALB/c mice but not in resistant C3H/HeJ and DBA/2 mice, an important splenomegaly. An in vitro defect in T-cell proliferation in response to T-cell receptor (TCR) stimulation with mitogens or anti-CD3 antibodies was associated with enhanced levels of CD4(+) and CD8(+) T-cell apoptosis in susceptible but not in resistant mice 2 weeks after infection. Further investigations of C57BL/6 and C3H/HeJ mice revealed that in vivo splenomegaly was associated with destruction of the lymphoid tissue architecture, liver cellular infiltrates, and increased numbers of apoptotic cells in both spleen and liver tissue sections. Infection of C57BL/6 mice but not of C3H/HeJ mice induced massive production of tumor necrosis factor alpha (TNF-alpha) in serum, as well as an increase in Fas and Fas ligand (FasL) expression in T cells. In vitro addition of neutralizing anti-TNF-alpha antibodies led to a significant reduction in CD3-induced T-cell apoptosis of both CD4(+) and CD8(+) T cells of C57BL/6 mice, while the blockade of Fas-FasL interactions reduced apoptosis only in CD4(+) but not in CD8(+) T cells. Together, these results suggest that TNF-alpha and Fas-FasL interactions play a role in the activation-induced cell death (AICD) process associated with a defect in T-cell proliferation of the susceptible C57BL/6 mice. T-cell death by apoptosis may represent one of the important components of the ineffective immune response against mycobacterium-induced immunopathology in susceptible hosts.

Animals↗
PubMed2000

New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis.

An in silico scan of the partially completed genome sequence of Bordetella pertussis and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to Bordetella virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.

Amino Acid Sequence↗