PubMed Health⌕ Search

Biomedical subjects

C Locht

Publications and source records attributed to C Locht.

At least 73 records · Page 4Linked to original sources
PubMed1997

Identification of novel intergenic repetitive units in a mycobacterial two-component system operon.

Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two-component system, named senX3-regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77-101 bp, 46-53 bp and 58-101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40-50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two-component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.

Amino Acid Sequence↗
PubMed1997

Immunodominant domains present on the Bordetella pertussis vaccine component filamentous hemagglutinin.

To identify immunologically important domains on filamentous hemagglutinin (FHA), a Bordetella pertussis protein included in new acellular pertussis vaccines (ACPVs), a series of monoclonal antibodies, sera from infants vaccinated with ACPVs or whole cell pertussis vaccine (WCPV), and sera from patients with pertussis were analyzed by immunoblots containing FHA fragments and recombinant FHA proteins. Immunodominant domains located at the COOH-terminus of FHA (type I domain) and near the NH2-terminus (type II domain) were defined by the reactivity with monoclonal antibodies. The sera from patients with pertussis and sera from infants vaccinated with WCPV or with 6 different investigational ACPVs specifically recognized well-defined regions within the type I and type II domains. Identification of these prominent immunologic epitopes on FHA should be useful for the construction of more well-defined pertussis vaccines and for the interpretation of human serologic responses, which may correlate with efficacy of pertussis vaccines.

Adhesins, Bacterial↗
PubMed1997

Intranasal priming with recombinant Bordetella pertussis for the induction of a systemic immune response against a heterologous antigen.

One of the current goals in vaccine development is the noninvasive administration of protective antigens via mucosal surfaces. In this context, the gut-associated lymphoid tissues have already been extensively explored. Vaccination via the nasal route has only recently been the focus of intensive investigation, and no live vector specifically designed for the respiratory mucosa is yet available. In this study we show that intranasal administration of the recombinant Bordetella pertussis BPGR60, producing the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28GST) protective antigen fused to filamentous hemagglutinin, induces priming in mice for the production of serum antibodies. In addition to significant levels of anti-Sm28GST immunoglobulin A (IgA) antibodies, high levels of anti-Sm28GST serum antibodies were obtained after intranasal boost with the purified antigen or infection with S. mansoni following intranasal priming with BPGR60. These antibodies were of the IgG1, IgG2a, and IgG2b isotypes, suggesting a mixed immune response. No priming was observed in animals that had received nonrecombinant B. pertussis or purified Sm28GST, indicating specific priming by BPGR60. This priming was also evident in immune protection against S. mansoni challenge. Significant protection against worm burden and egg output was obtained in mice primed with BPGR60 and intranasally boosted with purified Sm28GST. A lower but still significant degree of protection against egg output was also obtained in mice infected with a single dose of BPGR60. These results indicate that intranasal administration of recombinant B. pertussis can prime for serum antibody responses against a foreign antigen and for heterologous protection.

Administration, Intranasal↗
PubMed1997

Lack of functional complementation between Bordetella pertussis filamentous hemagglutinin and Proteus mirabilis HpmA hemolysin secretion machineries.

The gram-negative bacterium Bordetella pertussis has adapted specific secretion machineries for each of its major secretory proteins. In particular, the highly efficient secretion of filamentous hemagglutinin (FHA) is mediated by the accessory protein FhaC. FhaC belongs to a family of outer membrane proteins which are involved in the secretion of large adhesins or in the activation and secretion of Ca2+-independent hemolysins by several gram-negative bacteria. FHA shares with these hemolysins a 115-residue-long amino-proximal region essential for its secretion. To compare the secretory pathways of these hemolysins and FHA, we attempted functional transcomplementation between FhaC and the Proteus mirabilis hemolysin accessory protein HpmB. HpmB could not promote the secretion of FHA derivatives. Likewise, FhaC proved to be unable to mediate secretion and activation of HpmA, the cognate secretory partner of HpmB. In contrast, ShlB, the accessory protein of the closely related Serratia marcescens hemolysin, was able to activate and secrete HpmA. Two invariant asparagine residues lying in the region of homology shared by secretory proteins and shown to be essential for the secretion and activation of the hemolysins were replaced in FHA by site-directed mutagenesis. Replacements of these residues indicated that both are involved in, but only the first one is crucial to, FHA secretion. This slight discrepancy together with the lack of functional complementation demonstrates major differences between the hemolysins and FHA secretion machineries.

Adhesins, Bacterial↗
PubMed1996

A new series of mycobacterial expression vectors for the development of live recombinant vaccines.

Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.

Antigens↗
PubMed1996

Identification of a heparin-binding hemagglutinin present in mycobacteria.

Adherence to mammalian host tissues is an important virulence trait in microbial pathogenesis, yet little is known about the adherence mechanisms of mycobacteria. Here, we show that binding of mycobacteria to epithelial cells but not to macrophages can be specifically inhibited by sulfated carbohydrates. Using heparin-Sepharose chromatography, a 28-kD heparin-binding protein was purified from culture supernatants and cell extracts of Mycobacterium bovis and Mycobacterium tuberculosis. This protein, designated heparin-binding hemagglutinin (HBHA), promotes the agglutination of rabbit erythrocytes, which is specifically inhibited by sulfated carbohydrates. HBHA also induce mycobacterial aggregation, suggesting that it can mediate bacteria-bacteria interactions as well. Hemagglutination, mycobacterial aggregation, as well as attachment to epithelial cells are specifically inhibited in the presence of anti-HBHA antibodies. Immunoelectron microscopy using anti-HBHA monoclonal antibodies revealed that the protein is surface exposed, consistent with a role in adherence. Immunoblot analyses using antigen-specific antibodies indicated that HBHA is different from the fibronectin-binding proteins of the antigen 85 complex and p55, and comparison of the NH2-terminal amino acid sequence of purified HBHA with the protein sequence data bases did not reveal any significant similarity with other known proteins. Sera from tuberculosis patients but not from healthy individuals were found to recognize HBHA, indicating its immunogenicity in humans during mycobacterial infections. Identification of putative mycobacterial adhesins, such as the one described in this report, may provide the basis for the development of new therapeutic and prophylactic strategies against mycobacterial diseases.

Animals↗
PubMed1996

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Adhesins, Bacterial↗
PubMed1996

Neutralizing antibody responses elicited in mice immunized with recombinant bacillus Calmette-Guérin producing the Schistosoma mansoni glutathione S-transferase.

Schistosomiasis is a group of severe parasitic diseases, in humans and domestic animals, that are especially of importance in the developing world. No efficacious vaccine is currently available. However, Ab-mediated immune responses against the 28-kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST) appear to be involved in protection. This Ag was produced in recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG). The recombinant protein bound glutathione and expressed enzymatic activity, indicating that the active site of Sm28GST was folded properly. Single i.v., i.p., s.c., or intranasal immunizations with rBCG in BALB/c mice resulted in significant anti-SM28GST Ab responses, which were enhanced by a booster dose. The Ab responses remained high for at least 1 yr after immunization. Analyses of the isotype profiles indicated that i.v. immunized mice produced high titers of anti-Sm28GST IgG2a, and less IgG2b and IgG1. Mice immunized by the s.c. route initially also produced high levels of IgG2a and low titers of IgG1 and IgG2b, but the titers of the latter two isotypes increased gradually thereafter, tending toward a mixed profile. Intraperitoneal immunization provided a mixed profile directly after the first administration. High titers of anti-Sm28GST Abs also corresponded to high levels of neutralization of the enzymatic activity. These results indicate that rBCG induces strong IgG1, IgG2a, and IgG2b, and neutralizing Ab responses against Sm28GST, which has been found to correlate with protection against S. mansoni in humans.

Animals↗
PubMed1996

Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin.

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.

Alkaline Phosphatase↗
PubMed1996

Efficient homologous recombination in fast-growing and slow-growing mycobacteria.

Although homologous recombination is a major mechanism for DNA rearrangement in most living organisms, it has been difficult to detect in slowly growing mycobacteria by a classical suicide vector approach. Among the possible reasons for this are the low levels of transformation efficiency, the relatively high levels of illegitimate recombination, and the peculiar nature of the recA gene in slowly growing mycobacteria. In this report, we present an efficient homologous recombination system for these organisms based on the use of replicative plasmids which facilitates the detection of rare recombination events, because the proportions of recombined molecules increase over time. Intraplasmid homologous recombination in Mycobacterium smegmatis and Mycobacterium bovis BCG was easily selected by the reconstitution of an interrupted kanamycin resistance gene. Chromosomal integration via homologous recombination was selected by the expression of the kanamycin resistance gene under the control of a chromosomal promoter that was not present in the plasmid before recombination. This technique was termed STORE (for selection technique of recombination events). All the clones selected by STORE had undergone homologous recombination, as evidenced by PCR analyses of the kanamycin-resistant clones. This technique should be applicable to all organisms for which homologous recombination has been difficult to achieve, provided the gene of interest is expressed.

Amino Acid Sequence↗
PubMed1996

Distinct roles of the N-terminal and C-terminal precursor domains in the biogenesis of the Bordetella pertussis filamentous hemagglutinin.

The 220-kDa Bordetella pertussis filamentous hemagglutinin (FHA) is the major exported protein found in culture supernatants. The structural gene of FHA has a coding potential for a 367-kDa protein, and the mature form constitutes the N-terminal 60% of the 367-kDa precursor. The C-terminal domain of the precursor was found to be important for the high-level secretion of full-length FHA but not of truncated analogs (80 kDa or less). The secretion of full-length and truncated FHA polypeptides requires the presence of the approximately 100-amino-acid N-terminal domain and the outer membrane protein FhaC, homologous to the N-terminal domains of the Serratia marcescens and Proteus mirabilis hemolysins and their accessory proteins, respectively. By analogy to these hemolysins, it is likely that the N-terminal domain of the FHA precursor interacts, directly or indirectly, with the accessory protein during FHA biogenesis. However, immunogenicity and antigenicity studies suggest that the N-terminal domain of FHA is masked by its C-terminal domain and therefore should not be available for its interactions with FhaC. These observations suggest a model in which the C-terminal domain of the FHA precursor may play a role as an intramolecular chaperone to prevent premature folding of the protein. Both heparin binding and hemagglutination are expressed by the N-terminal half of FHA, indicating that this domain contains important functional regions of the molecule.

Adhesins, Bacterial↗
PubMed1996

Immune system-dependent and -independent replication of the scrapie agent.

Using the severe combined immunodeficiency (SCID) mouse model, we investigated the requirement of the immune system for the development of scrapie after peripheral inoculation. A total of 33% of SCID mice, all but one immunologically reconstituted SCID mice (93%), and all CB17 control mice developed the disease. PrPres was detectable in the brains of all diseased animals and in the spleens of reconstituted SCID and CB17 control mice but not of the diseased non-immunologically reconstituted SCID mice. The immune system appears to be a primary target in the pathogenesis of scrapie, but direct spread to the central nervous system from the peritoneum via visceral nerve fibers can probably also occur.

Animals↗
PubMed1995

A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit.

Pertussis toxin is a complex protein composed of five different subunits, named S1 through S5 and arranged in an A-B structure. The B oligomer, composed of S2 through S5, is the receptor-binding moiety, and the A promoter, composed of S1, is the enzymatically active moiety. S1 catalyzes the ADP-ribosylation of a cysteine in the alpha subunit of heterotrimeric G proteins. In the absence of G proteins it also catalyzes the cleavage of NAD+ into ADP-ribose and nicotinamide. Molecular dissection has indicated that the C-terminal domain of S1 is involved in G-protein binding, while the N-terminal domain, homologous to other ADP-ribosylating toxins, contains the NAD(+)-binding site and the residues involved in catalysis. By site-directed mutagenesis and kinetic analyses Glu-129 and His-35 were identified as the catalytic residues. Glutamates analogous to Glu-129 are found in all studied ADP-ribosylating toxins, while His-35 is less well conserved. This suggests that Glu-129 acts on the common substrate NAD+, whereas His-35 plays its role on the acceptor substrates. We propose a mechanism in which Glu-129 exerts its action on the 2'-OH group of the NAD+ ribose, thereby facilitating the formation of an oxocarbonium-like intermediate and the weakening of the N-glycosidic bond. His-35 could increase the nucleophilicity of the cysteine in the G protein or the water molecule to attack the weakened N-glycosidic bond of NAD+ and yield the products of the reaction.

Adenosine Diphosphate Ribose↗
PubMed1995

Mercury resistance as a selective marker for recombinant mycobacteria.

The use of antibiotic-resistance markers for the selection of recombinant mycobacteria is widespread but questionable considering the development of live recombinant BCG vaccines. In contrast, vector-encoded resistance to heavy metals such as mercury may represent an interesting alternative for the development of live vaccines compatible with use in humans and in animals. The mercury resistance genes (mer) from Pseudomonas aeruginosa and from Serratia marcescens were cloned into the Escherichia coli-Mycobacterium shuttle vector pRR3. The resulting vectors, designated pMR001 and pVN2, were introduced by electroporation into Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis. The recombinant mycobacteria were stable in vitro and in vivo, and had high-level mercury resistance, thus indicating that the mer genes can be useful as selective markers in mycobacteria.

Animals↗
PubMed1995

Green fluorescent protein as a new expression marker in mycobacteria.

This study describes the use and the advantages of the green fluorescent protein (GFP) as a reporter molecule for mycobacteria. The gfp gene from Aequorea victoria was placed under the control of the hsp60 promoter in the shuttle vector pGFM-11. The gfp expression in the recombinant Mycobacterium smegmatis and BCG was readily detected on agar plates by the development of an intense green fluorescence upon irradiation with long-wave u.v. light. In mycobacteria containing a pGFM-11 derivative that lacks the hsp60 promoter, no fluorescence was observed. However, this plasmid was successfully used as a promoter-probe vector to identify BCG promoters. The fluorescence emission of GFP in mycobacteria harbouring pGFM-11 and grown in liquid media could be quantified by spectrofluorimetry. This allowed for easy assessment of drug susceptibility. As GFP does not require the addition of substrates or co-factors, the green fluorescent bacilli could be directly observed within infected macrophages using fluorescence and laser confocal microscopy, or in tissue sections of infected mice. Finally, infected cells or free-living recombinant mycobacteria could also be analysed by flow cytometry. The GFP thus appears to be a convenient reporter for mycobacteria, allowing tracing of recombinant mycobacteria, isolation of promoters with interesting properties, in vivo drug testing and the development of new diagnostic tools.

Amino Acid Sequence↗
PubMed1995

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Alkaline Phosphatase↗
PubMed1994

The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis.

Control of virulence factor expression in Bordetella pertussis is mediated by the products of the bvg operon. The BvgS membrane protein responds to certain environmental cues by activating the BvgA protein, which in turn modulates the expression of the target virulence factor genes. The BvgA and BvgS proteins are members of a large family of sensory transduction proteins called the two-component systems. We show that BvgA fusion proteins can activate transcription of a reporter gene containing the bvg promoter in Escherichia coli, and that this activity correlates with its ability to interact specifically with a recognition sequence in cognate promoters. Using homologies between BvgA and other bacterial response regulators as a guide, two BvgA truncation mutants were constructed and their transactivation and DNA-binding capacities were examined. We discovered that (1) DNA-binding activity is localized to the C-terminal half of BvgA, (2) sequence-specific DNA-binding is necessary, but not sufficient for transactivation, and (3) DNA-binding requires the last 20 amino acid residues at its carboxy terminus. A BvgA fusion protein lacking the receiver domain is inactive in transcriptional activation, but retains sequence-specific DNA-binding activity and forms multimeric complexes. We show that BvgA is able to utilize acetyl phosphate as a phosphoryl group donor and the instability of the covalent linkage at extremes of pH is consistent with an acyl phosphate group. Furthermore, the in vitro phosphorylated form of BvgA exhibits an enhanced capacity for binding DNA target sites, while a dephosphorylated form exhibits a limited capacity to bind these sites. We discuss the implications that these observations have on the mechanism by which BvgA is activated to a transcriptionally competent state.

Bacterial Proteins↗