PubMed Health⌕ Search

Biomedical subjects

Xavier De Bolle

Publications and source records attributed to Xavier De Bolle.

17 recordsLinked to original sources
PubMed2006

Gateway-based destination vectors for functional analyses of bacterial ORFeomes: application to the Min system in Brucella abortus.

Twenty Gateway-compatible destination vectors were constructed. The vectors comprise fluorescent and epitope fusion tags, various drug markers, and replication origins that should make them useful for exploring existing microbial ORFeomes. In an attempt to validate several of these vectors, we observed polar and oscillating localization of MinD in Brucella abortus.

Brucella abortus↗
PubMed2006

An RpoH-like heat shock sigma factor is involved in stress response and virulence in Brucella melitensis 16M.

B. melitensis 16M genome analysis revealed the presence of six putative sigma factor-encoding genes: rpoD, rpoH1, rpoH2, rpoE1, rpoE2, and rpoN. We mutated all these genes except rpoD. Phenotypic analysis of the mutants reveals that a strain carrying an rpoH2 null mutation (DeltarpoH2) is impaired for growth at 21 and 42 degrees C and shows increased sensitivity to hydrogen peroxide. Compared to the wild-type strain, the DeltarpoH2 mutant is attenuated in all virulence models tested. Three other null mutants (DeltarpoH1, DeltarpoE1, and DeltarpoE2 mutants) are also defective for survival in mice at 4 weeks postinfection. We also demonstrated that rpoH2 deletion strongly reduces the expression of two major virulence factors in B. melitensis, the type IV secretion system and the flagellum.

Adaptation, Physiological↗
PubMed2006

The stringent response mediator Rsh is required for Brucella melitensis and Brucella suis virulence, and for expression of the type IV secretion system virB.

Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, possesses a gene homologous to relA/spoT, named rsh, encoding a (p)ppGpp synthetase as confirmed by heterologous complementation of a relA mutant of Sinorhizobium meliloti. The Rsh deletion mutants in Brucella suis and Brucella melitensis were characterized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evidenced that expression of virB, encoding the type IV secretion system, a major virulence factor of Brucella, was Rsh-dependent. All mutant phenotypes, including lack of VirB proteins, were complemented with the rsh gene of Brucella. In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella, Rsh might participate in the adaptation of the pathogen to low-nutrient environments, and indirectly in the VirB-mediated formation of the final replicative niche.

Animals↗
PubMed2005

A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis.

Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C(12)-homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella.

4-Butyrolactone↗
PubMed2005

Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence.

In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.

Animals↗
PubMed2004

Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex.

BACKGROUND: Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). METHODS: We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing. RESULTS: Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL. CONCLUSION: Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.

Animals↗
PubMed2004

Brucella pathogenesis, genes identified from random large-scale screens.

Pathogenicity islands, specialized secretion systems, virulence plasmids, fimbriae, pili, adhesins, and toxins are all classical bacterial virulence factors. However, many of these factors, though widespread among bacterial pathogens, are not necessarily found among bacteria that colonize eukaryotic cells in a pathogenic/symbiotic relationship. Bacteria that form these relationships have developed other strategies to infect and grow in their hosts. This is particularly true for Brucella and other members of the class Proteobacteria. Thus far the identification of virulence factors for Brucella has been largely dependent on large-scale screens and testing in model systems. The genomes of the facultative intracellular pathogens Brucella melitensis and Brucella suis were sequenced recently. This has identified several more potential virulence factors for Brucella that were not found in large screens. Here, we present an overall view of Brucella virulence by compiling virulence data from the study of 184 attenuated mutants.

Animals↗
PubMed2004

Functional, molecular and structural characterisation of five anti-Brucella LPS mAb.

The O-antigen of the gram negative bacteria Brucella is composed of an homopolymer of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl (or perosamine). Several mAb interact specifically with only the O-antigen of certain Brucella species. Although, many studies show that this specific recognition results mainly from the ratios of alpha 1-2 and alpha 1-3 link between the different Brucella strain perosamine residues, little is known about the mAb recognising this O-antigen. In this paper, we describe the binding profile of five anti-Brucella O-antigen mAb to the LPS of two Brucella strains and a bacteria possessing a nearly identical O-antigen: Yersinia enterocolitica 0:9. We show that the specificity of these five mAb can be correlated to their germ line gene usage. Besides, their relative affinity to the different LPS is correlated to their ability to protect against Brucella infection by passive transfer in a mouse model. The analysis of their 3D structure gives new hypothesis of the epitopes recognised.

Amino Acid Sequence↗
PubMed2004

Morphological and functional asymmetry in alpha-proteobacteria.

The release of an increasing number of complete bacterial genomic sequences allows the evolutionary analysis of processes such as regulatory networks. CtrA is a response regulator of the OmpR subfamily, belonging to a complex regulatory network in the dimorphic bacterium Caulobacter crescentus. It coordinates the cell cycle with an asymmetric division, which is part of the adaptation of Caulobacter to poor-nutrient environments. CtrA is only found in alpha-proteobacteria, a group of bacteria encompassing genera with very distinct lifestyles, including host-associated bacteria. Analyses of CtrA regulatory networks and morphological examinations of some alpha-proteobacteria are presented. Our observations suggest that the core of the CtrA regulation network is conserved and that alpha-proteobacteria divide asymmetrically. We propose that the two daughter cells might be differentiated bacteria, each one displaying specific functions.

Bacterial Proteins↗
PubMed2004

Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Bacterial Proteins↗
PubMed2004

The Ton system, an ABC transporter, and a universally conserved GTPase are involved in iron utilization by Brucella melitensis 16M.

Brucella spp. are gram-negative intracellular facultative pathogens that are known to produce 2,3-dihydroxybenzoic acid (DHBA), a catechol siderophore that is essential for full virulence in the natural host. The mechanism of DHBA entry into Brucella and other gram-negative bacteria is poorly understood. Using mini-Tn5Kmcat mutagenesis, we created a transposon library of Brucella melitensis 16M and isolated 32 mutants with a defect in iron acquisition or assimilation. Three of these transposon mutants are deficient in utilization of DHBA. Analysis of these three mutants indicated that the ExbB, DstC, and DugA proteins are required for optimal assimilation of DHBA and/or citrate. ExbB is part of the Ton complex, and DstC is a permease homologue of an iron(III) ABC transporter; in gram-negative bacteria these two complexes are involved in the uptake of iron through the outer and inner membranes, respectively. DugA is a new partner in iron utilization that exhibits homology with the bacterial conserved GTPase YchF. Based on this homology, DugA could have a putative regulatory function in iron assimilation in Brucella. None of the three mutants was attenuated in cellular models or in the mouse model of infection, which is consistent with the previous suggestion that DHBA utilization is not required in these models.

ATP-Binding Cassette Transporters↗
PubMed2003

Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning.

The Gateway technology is becoming an increasingly popular method for cloning ORFs by recombination. It allows the transfer of any ORF flanked by specific recombination sites into any vectors harbouring the corresponding sites. Here we describe the construction of a set of 20 Saccharomyces cerevisiae Gateway compatible vectors. These plasmids bear an URA3 or TRP1 selection marker. They are designed for expression without tag sequence or for C- or N-terminal protein tagging with 3HA (haemagglutinin), 13MYC, 4TAP (tandem affinity purification) or GST (glutathione S-transferase) epitopes. The centromeric vectors allow expression of DNA sequence in yeast under tetracycline-regulatable promoters, while expression from the high copy vectors is driven by PGK promoter. To test their applicability, the genes encoding the RNA polymerase I subunit Rpa12p or the TFIIS transcription factor were cloned in these vectors. Their expression was demonstrated using Western blotting or complementation assays.

Cloning, Molecular↗
PubMed2002

Plasticity of a transcriptional regulation network among alpha-proteobacteria is supported by the identification of CtrA targets in Brucella abortus.

CtrA is a master response regulator found in many alpha-proteobacteria. In Caulobacter crescentus and Sinorhizobium meliloti, this regulator is essential for viability and is transcriptionally autoregulated. In C. crescentus, it is required for the regulation of multiple cell cycle events, such as DNA methylation, DNA replication, flagella and pili biogenesis and septation. Here, we report the characterization of the ctrA gene homologue in the alpha2-proteobacteria Brucella abortus, a facultative intracellular pathogen responsible for brucellosis. We detected CtrA expression in the main Brucella species, and its overproduction led to a phenotype typical of cell division defect, consistent with its expected role. A purified B. abortus CtrA recombinant protein (His6-CtrA) was shown to protect the B. abortus ctrA promoter from DNase I digestion, suggesting transcriptional autoregulation, and this protection was enhanced under CtrA phosphorylation on a conserved Asp residue. Despite the similarities shared by B. abortus and C. crescentus ctrA, the pathway downstream from CtrA may be distinct, at least partially, in both bacteria. Indeed, beside ctrA itself, only one (the ccrM gene) out of four B. abortus homologues of known C. crescentus CtrA targets is bound in vitro by phosphorylated B. abortus CtrA. Moreover, further footprinting experiments support the hypothesis that, in B. abortus, CtrA might directly regulate the expression of the rpoD, pleC, minC and ftsE homologues. Taken together, these results suggest that, in B. abortus and C. crescentus, similar cellular processes are regulated by CtrA through the control of distinct target genes. The plasticity of the regulation network involving CtrA in these two bacteria may be related to their distinct lifestyles.

Alphaproteobacteria↗
PubMed2002

ESyPred3D: Prediction of proteins 3D structures.

MOTIVATION: Homology or comparative modeling is currently the most accurate method to predict the three-dimensional structure of proteins. It generally consists in four steps: (1) databanks searching to identify the structural homolog, (2) target-template alignment, (3) model building and optimization, and (4) model evaluation. The target-template alignment step is generally accepted as the most critical step in homology modeling. RESULTS: We present here ESyPred3D, a new automated homology modeling program. The method gets benefit of the increased alignment performances of a new alignment strategy. Alignments are obtained by combining, weighting and screening the results of several multiple alignment programs. The final three-dimensional structure is build using the modeling package MODELLER. ESyPred3D was tested on 13 targets in the CASP4 experiment (Critical Assessment of Techniques for Proteins Structural Prediction). Our alignment strategy obtains better results compared to PSI-BLAST alignments and ESyPred3D alignments are among the most accurate compared to those of participants having used the same template. AVAILABILITY: ESyPred3D is available through its web site at http://www.fundp.ac.be/urbm/bioinfo/esypred/ CONTACT: christophe.lambert@fundp.ac.be; http://www.fundp.ac.be/~lambertc

Algorithms↗
PubMed2002

Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen.

Brucella is a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response against Brucella involves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominant Brucella antigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuated Yersinia enterocolitica serotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, the Yersinia vectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizing Brucella abortus 544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection by B. abortus 544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains of Yersinia O:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity against B. abortus 544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism.

Animals↗
PubMed2002

Identification of a quorum-sensing signal molecule in the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

4-Butyrolactone↗