Biomedical subjects

X De Bolle

Publications and source records attributed to X De Bolle.

15 recordsLinked to original sources

PubMed2001

Identification of Brucella spp. genes involved in intracellular trafficking.

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago-lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis. We also examined the intracellular fate of three virB mutants (virB2, virB4 and virB9) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago-lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61beta protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.

Bacterial Proteins↗

PubMed2001

Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp.

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins of Brucella spp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge with B. abortus 544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens or Brucella extract. In this report, we also demonstrate that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged with B. abortus 544 compared to that observed in positive control mice vaccinated with S19.

Animals↗

PubMed2001

Protection of BALB/c mice against Brucella abortus 544 challenge by vaccination with bacterioferritin or P39 recombinant proteins with CpG oligodeoxynucleotides as adjuvant.

The P39 and the bacterioferrin (BFR) antigens of Brucella melitensis 16M were previously identified as T dominant antigens able to induce both delayed-type hypersensivity in sensitized guinea pigs and in vitro gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells from infected cattle. Here, we analyzed the potential for these antigens to function as a subunitary vaccine against Brucella abortus infection in BALB/c mice, and we characterized the humoral and cellular immune responses induced. Mice were injected with each of the recombinant proteins alone or adjuvanted with either CpG oligodeoxynucleotides (CpG ODN) or non-CpG ODN. Mice immunized with the recombinant antigens with CpG ODN were the only group demonstrating both significant IFN-gamma production and T-cell proliferation in response to either Brucella extract or to the respective antigen. The same conclusion holds true for the antibody response, which was only demonstrated in mice immunized with recombinant antigens mixed with CpG ODN. The antibody titers (both immunoglobulin G1 [IgG1] and IgG2a) induced by P39 immunization were higher than the titers induced by BFR (only IgG2a). Using a B. abortus 544 challenge, the level of protection was analyzed and compared to the protection conferred by one immunization with the vaccine strain B19. Immunization with P39 and CpG ODN gave a level of protection comparable to the one conferred by B19 at 4 weeks postchallenge, and the mice were still significantly protected at 8 weeks postchallenge, although to a lesser extent than the B19-vaccinated group. Intriguingly, no protection was detected after BFR vaccination. All other groups did not demonstrate any protection.

Adjuvants, Immunologic↗

PubMed2000

The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases.

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5'-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17-38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.

Base Sequence↗

PubMed2000

Identification and characterization of in vivo attenuated mutants of Brucella melitensis.

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

Animals↗

PubMed1999

Antigenic properties of peptidic mimics for epitopes of the lipopolysaccharide from Brucella.

The lipopolysaccharide (LPS) is up to now the only identified major virulence determinant of Brucella. This bacterium is responsible for brucellosis in animals and for Malta fever in humans. Several monoclonal antibodies (mAbs) directed against various LPS epitopes have been characterized. Two mAbs, named A15-6B3 and B66-2C8, directed against distinct LPS epitopes have been used to select peptides from 11 phage display libraries. The sequences of the selected peptides contain an overrepresentation of either proline or tryptophan residues when selected with either A15-6B3 or B66-2C8 mAbs, respectively. For the best binding peptides, competition with LPS for the binding to the mAb is detected, which suggests that the peptides bind to the paratope of the mAb. The phages selected from the libraries were used to immunise mice, and a weak antibody response directed against LPS has been observed. These data suggest that a subset of the selected peptides are mimotopes of the LPS epitopes.

Amino Acid Sequence↗

PubMed1999

Structure and function prediction of the Brucella abortus P39 protein by comparative modeling with marginal sequence similarities.

A methodology is proposed to solve a difficult modeling problem related to the recently sequenced P39 protein. This sequence shares no similarity with any known 3D structure, but a fold is proposed by several threading tools. The difficulty in aligning the target sequence on one of the proposed template structures is overcome by combining the results of several available prediction methods and by refining a rational consensus between them. In silico validation of the obtained model and a preliminary cross-check with experimental features allow us to state that this borderline prediction is at least reasonable. This model raises relevant hypotheses on the main structural features of the protein and allows the design of site-directed mutations. Knowing the genetic context of the P39 reading frame, we are now able to suggest a function for the P39 protein: it would act as a periplasmic substrate-binding protein.

Amino Acid Sequence↗

PubMed1998

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4.

Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.

Animals↗

PubMed1998

Comparative analysis of seven multiple protein sequence alignment servers: clues to enhance reliability of predictions.

MOTIVATION: The prediction reliability of seven multiple alignment servers currently available on the Internet (ClustalW, MAP, PIMA, Block Maker, MSA, MEME and Match-Box) has been evaluated in terms of power (sensitivity) and confidence (selectivity). Therefore, the alignments obtained have been respectively compared to refined structural alignments for 20 families of related proteins with low levels of identity. RESULTS: Results clearly show that any powerful method remains reliable when the rate of identity falls. For some methods, power and confidence decrease linearly with the rate of identity, while other methods emphasize reliability at the cost of a lower power. Increasing the number of related sequences included in the alignment may either improve or decrease the quality of the predictions substantially. For some methods, the gain in power or in confidence is quite systematic; for others, the effect of the addition of homologous sequences is highly unpredictable. Extracting the consensus between two different methods may increase the overall confidence of the predictions tremendously. Our conclusions induce users of sequence alignment methods on the Internet to select the most suitable technique according to their requirements in terms of selectivity and sensitivity. AVAILABILITY: The aligned sequences of the 20 alignments of structure can be obtained automatically by sending the message 'send: cabios_tests.txt' by e-mail to 'matchbox@biq.fundp.ac.be'. CONTACT: eric.depiereux@fundp.ac.be

Computer Communication Networks↗

PubMed1997

Bivalent cations stabilize yeast alcohol dehydrogenase I.

The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme. Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations. Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme. An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure. These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation. It is proposed that yeast ADH I binds Mg2+ in vivo.

Alcohol Dehydrogenase↗

PubMed1997

Match-Box_server: a multiple sequence alignment tool placing emphasis on reliability.

MOTIVATION: The Match-Box software comprises protein sequence alignment tools based on strict statistical thresholds of similarity between protein segments. The method circumvents the gap penalty requirement: gaps being the result of the alignment and not a governing parameter of the procedure. The reliable conserved regions outlined by Match-Box are particularly relevant for homology modelling of protein structures, prediction of essential residues for site-directed mutagenesis and oligonucleotide design for cloning homologous genes by polymerase chain reaction (PCR). RESULTS: The method produces reliable results, as assessed by tests performed on protein families of known structures and of low sequence similarity. A reliability score is computed in relation to a threshold of similarity progressively raised to extend the aligned regions to their maximal length, up to the significance limit of matching segments. The score obtained at each position is printed below the sequences and allows a discriminant reading of each aligned region. AVAILABILITY: Sequences may be submitted to a Web server at http://www.fundp.ac.be/sciences/biologie/bms/+ ++matchbox_submit.html or sent by e-mail to matchbox/biq.fundp.ac.be (help available by just mailing help).

Algorithms↗

PubMed1995

Identification of residues potentially involved in the interactions between subunits in yeast alcohol dehydrogenases.

The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH II isozymes as well as of several chimeric (ADH I-ADH II) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and II. The first group contains a Cys277-->Ser substitution, located at the interface between subunits in a three-dimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-->Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Met168-->Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.

Alcohol Dehydrogenase↗

PubMed1995

Knowledge-based modeling of the D-lactate dehydrogenase three-dimensional structure.

A three-dimensional structure of the NAD-dependent D-lactate dehydrogenase of Lactobacillus bulgaricus is modeled using the structure of the formate dehydrogenase of Pseudomonas sp. as template. Both sequences share only 22% of identical residues. Regions for knowledge-based modeling are defined from the structurally conserved regions predicted by multiple alignment of a set of related protein sequences with low homology. The model of the D-LDH subunit shows, as for the formate dehydrogenase, an alpha/beta structure, with a catalytic domain and a coenzyme binding domain. It points out the catalytic histidine (His-296) and supports the hypothetical catalytic mechanism. It also suggests that the other residues involved in the active site are Arg-235, possibly involved in the binding of the carboxyl group of the pyruvate, and Phe-299, a candidate for stabilizing the methyl group of the substrate.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins.

Recombinant lambda gt11 phages were selected by screening a genomic library of Brucella abortus DNA with monoclonal antibodies specific for a 16.5-kDa Brucella outer membrane protein (Omp16). The corresponding gene, named pal, was subcloned on a 0.7-kb AluI fragment. Immunoblotting confirmed the expression of a recombinant Omp16 in the transformants. DNA sequence analysis revealed an open reading frame of 168 codons. The deduced amino acid sequence agrees with an internal peptide sequence of native Omp16 and contains a potential lipoprotein signal peptide cleavage site, giving rise to a predicted mature protein of 144 amino acids. The predicted sequence of Omp16 also shows a remarkable degree of similarity to the sequences of three peptidoglycan-associated bacterial lipoproteins. In immunoblotting with a monoclonal antibody specific for Omp16, we demonstrated that Omp16 was expressed in the 34 Brucella strains tested, representing all six species and known biovars.

Amino Acid Sequence↗

PubMed1993

Similarities between alanine dehydrogenase and the N-terminal part of pyridine nucleotide transhydrogenase and their possible implication in the virulence mechanism of Mycobacterium tuberculosis.

Recent developments in simultaneous multiple alignment methods of protein sequences allow prediction of structural similarity in related proteins. Alanine dehydrogenase and the N-terminal sequence of pyridine nucleotide transhydrogenase were compared for their sequences. High similarities of sequences were observed especially in their NAD(H)-binding sites. These similarities suggest that antibodies which recognized the alanine dehydrogenase of Mycobacterium tuberculosis can also be directed against the membrane bound pyridine nucleotide transhydrogenase. If this is the case, the virulent property of this pathogen could be linked to its higher synthesis of NADPH necessary for its anabolism.

Alanine Dehydrogenase↗