Biomedical subjects

Remy Loris

Publications and source records attributed to Remy Loris.

At least 19 recordsLinked to original sources

PubMed2025

Isolation and characterization of bacteriophages from clinical enterohemorrhagic Escherichia coli strains.

Temperate bacteriophages play a pivotal role in the biology of their bacterial host. Of particular interest are bacteriophages infecting enterohemorrhagic E. coli (EHEC) due to their significant contribution to the pathogenicity of its host, most notably by encoding the key virulence factor of this pathogen, the Shiga toxin. To better understand the role of EHEC phages on the functionality of its host, we isolated eight temperate phages from clinical EHEC isolates and characterized their genomic composition, morphology, and receptor targeting. Morphological analysis identified one long-tailed siphophage, targeting the OmpC receptor for host recognition, whereas the other seven phages are short-tailed podophages and target the essential BamA protein. Genomic characterization revealed significant variations between the long- and short-tailed phages. Five of the eight isolated phages encode the potent Shiga toxin. Comparative analysis displays the typical lambdoid mosaicism, indicative of horizontal gene transfer driving evolution. These findings provide insights into the genetic and morphologic diversity and receptor specificity of EHEC phages, highlighting their role in the evolution and pathogenicity of clinical EHEC strains.IMPORTANCECharacterizing bacteriophages from clinical EHEC isolates is crucial in understanding the mechanisms underlying bacterial evolution and virulence. Despite the clinical relevance of EHEC bacteriophages, they remain underexplored, and particularly phage receptors are often not characterized. Studying temperate EHEC phages is essential in the development of strategies to address the global burden of these foodborne infections. Notably, identifying the phage receptors is critical in unraveling the specific interaction between phage and host. Knowledge of the phage receptors can provide insights into the mechanisms of phage infection, host range, and bacterial resistance and is fundamental in the design of targeted therapies like new antimicrobials, phage therapy, or prevention of those infections.

Humans↗

PubMed2005

Toxin-antitoxin modules as bacterial metabolic stress managers.

Bacterial genomes frequently contain operons that encode a toxin and its antidote. These 'toxin-antitoxin (TA) modules' have an important role in bacterial stress physiology and might form the basis of multidrug resistance. The toxins in TA modules act as gyrase poisons or stall the ribosome by mediating the cleavage of mRNA. The antidotes contain an N-terminal DNA-binding region of variable fold and a C-terminal toxin-inhibiting domain. When bound to toxin, the C-terminal domain adopts an extended conformation. In the absence of toxin, by contrast, this domain (and sometimes the whole antidote protein) remains unstructured, allowing its fast degradation by proteolysis. Under silent conditions the antidote inhibits the toxin and the toxin-antidote complex acts as a repressor for the TA operon, whereas under conditions of activation proteolytic degradation of the antidote outpaces its synthesis.

Antitoxins↗

PubMed2005

Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies.

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDR-H loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJmol(-1)), good expression level (5 mgl(-1) in E.coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity.

Amino Acid Sequence↗

PubMed2005

Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.

Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.

Adhesins, Bacterial↗

PubMed2005

Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a VH.

Heavy chain only antibodies of camelids bind their antigens with a single domain, the VHH, which acquired adaptations relative to classical VHs to function in the absence of a VL partner. Additional CDR loop conformations, outside the canonical loop structures of VHs, broaden the repertoire of the antigen-binding site. The combined effects of part of the CDR3 that folds over the "former" VL binding site and framework-2 mutations to more hydrophilic amino acids, enhance the solubility of VHH domains and prevent VL pairing. cAbAn33, a VHH domain specific for the carbohydrate moiety of the variant surface glycoprotein of trypanosomes, has a short CDR3 loop that does not cover the former VL binding site as well as a VH-specific Trp47 instead of the VHH-specific Gly47. Resurfacing its framework-2 region (mutations Tyr37Val, Glu44Gly and Arg45Leu) to mimic that of a human VH restores the VL binding capacity. In solution, the humanised VHH behaves as a soluble, monomeric entity, albeit with reduced thermodynamic stability and affinity for its antigen. Comparison of the crystal structures of cAbAn33 and its humanised derivative reveals steric hindrance exerted by VHH-specific residues Tyr37 and Arg45 that prevent the VL domain pairing, whereas Glu44 and Arg45 are key elements to avoid insolubility of the domain.

Amino Acid Sequence↗

PubMed2005

Crystallization and crystal manipulation of the Pterocarpus angolensis seed lectin.

The Man/Glc-specific legume lectin from the seeds of the African bloodwood tree (Pterocarpus angolensis) was crystallized in the presence of the disaccharide ligand Man(alpha1-3)ManMe. Small crystals initially appeared from a preliminary screen, but proved difficult to reproduce. The initial crystals were used to prepare microseeds, leading to a reproducible crystallization protocol. All attempts to obtain crystals directly of the ligand-free protein or of other carbohydrate complexes failed. However, the Man(alpha1-3)ManMe co-crystals withstand soaking with ten other carbohydrates known to bind to the lectin. Soaking for 15 min in 100 mM carbohydrate typically resulted in complete replacement of Man(alpha1-3)ManMe by the desired carbohydrate despite the involvement of lattice contacts at the binding site. Transferring the crystals for two weeks in carbohydrate-free artificial mother liquor resulted in the complete removal of the sugar from one of the two monomers in the asymmetric unit. Additional treatment of these crystals with 100 mM EDTA for two weeks resulted in removal of the structural calcium and manganese ions, which is accompanied by significant structural rearrangements of the loops that constitute the carbohydrate-binding site.

Crystallography, X-Ray↗

PubMed2005

Molecular basis of gyrase poisoning by the addiction toxin CcdB.

Gyrase is an ubiquitous bacterial enzyme that is responsible for disentangling DNA during DNA replication and transcription. It is the target of the toxin CcdB, a paradigm for plasmid addiction systems and related bacterial toxin-antitoxin systems. The crystal structure of CcdB and the dimerization domain of the A subunit of gyrase (GyrA14) dictates an open conformation for the catalytic domain of gyrase when CcdB is bound. The action of CcdB is one of a wedge that stabilizes a dead-end covalent gyrase:DNA adduct. Although CcdB and GyrA14 form a globally symmetric complex where the two 2-fold axes of both dimers align, the complex is asymmetric in its details. At the centre of the interaction site, the Trp99 pair of CcdB stacks with the Arg462 pair of GyrA14, explaining why the Arg462Cys mutation in the A subunit of gyrase confers resistance to CcdB. Overexpression of GyrA14 protects Escherichia coli cells against CcdB, mimicking the action of the antidote CcdA.

Bacterial Proteins↗

PubMed2005

Energetics of structural transitions of the addiction antitoxin MazE: is a programmed bacterial cell death dependent on the intrinsically flexible nature of the antitoxins?

The Escherichia coli mazEF addiction module plays a crucial role in the cell death program that is triggered under various stress conditions. It codes for the toxin MazF and the antitoxin MazE, which interferes with the lethal action of the toxin. To better understand the role of various conformations of MazE in bacterial life, its order-disorder transitions were monitored by differential scanning calorimetry, spectropolarimetry, and fluorimetry. The changes in spectral and thermodynamic properties accompanying MazE dimer denaturation can be described in terms of a compensating reversible process of the partial folding of the unstructured C-terminal half (high mean net charge, low mean hydrophobicity) and monomerization coupled with the partial unfolding of the structured N-terminal half (low mean net charge, high mean hydrophobicity). At pH<or=4.5 and T<50 degrees C, the unstructured polypeptide chains of the MazE dimer fold into (pre)molten globule-like conformations that thermally stabilize the dimeric form of the protein. The simulation based on the thermodynamic and structural information on various addiction modules suggests that both the conformational adaptability of the dimeric antitoxin form (binding to the toxins and DNA) and the reversible transformation to the more flexible monomeric form are essential for the regulation of bacterial cell life and death.

Antitoxins↗

PubMed2005

Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires.

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.

Amino Acid Sequence↗

PubMed2005

Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation.

Pseudomonas aeruginosa is an opportunistic pathogen which causes a variety of diseases, including respiratory tract infections in patients suffering from cystic fibrosis. Therapeutic treatment of P. aeruginosa infections is still very difficult because the bacteria exhibit high intrinsic resistance against a variety of different antibiotics and, in addition, form stable biofilms, e.g. in the human lung. Several virulence factors are produced by P. aeruginosa, among them the two lectins LecA and LecB, which exert different cytotoxic effects on respiratory epithelial cells and presumably facilitate bacterial adhesion to the airway mucosa. Here, the physiology has been studied of the lectin LecB, which binds specifically to L-fucose. A LecB-deficient P. aeruginosa mutant was shown to be impaired in biofilm formation when compared with the wild-type strain, suggesting an important role for LecB in this process. This result prompted an investigation of the subcellular localization of LecB by cell fractionation and subsequent immunoblotting. The results show that LecB is abundantly present in the bacterial outer-membrane fraction. It is further demonstrated that LecB could be released specifically by treatment of the outer-membrane fraction with p-nitrophenyl alpha-L-fucose, whereas treatment with D-galactose had no effect. In contrast, a LecB protein carrying the mutation D104A, which results in a defective sugar-binding site, was no longer detectable in the membrane fraction, suggesting that LecB binds to specific carbohydrate ligands located at the bacterial cell surface. Staining of biofilm cells using fluorescently labelled LecB confirmed the presence of these ligands.

Bacterial Outer Membrane Proteins↗

PubMed2004

Overexpression, purification and crystallization of bacteriocin LlpA from Pseudomonas sp. BW11M1.

LlpA is a bacteriocin produced by Pseudomonas sp. BW11M1 that shows remarkable similarity to a family of mannose-binding plant lectins. A His-tagged version of LlpA was recombinantly produced in Escherichia coli and purified to homogeneity. Single crystals were grown by vapour diffusion and belong to space group P2(1)2(1)2, with unit-cell parameters a = 150.5, b = 154.5, c = 34.2 A. The crystals diffract to at least 2.2 A using synchrotron radiation.

Bacteriocins↗

PubMed2004

Chemical basis for the affinity maturation of a camel single domain antibody.

Affinity maturation of classic antibodies supposedly proceeds through the pre-organization of the reactive germ line conformational isomer. It is less evident to foresee how this can be accomplished by camelid heavy-chain antibodies lacking light chains. Although these antibodies are subjected to somatic hypermutation, their antigen-binding fragment consists of a single domain with restricted flexibility in favor of binding energy. An antigen-binding domain derived from a dromedary heavy-chain antibody, cAb-Lys3, accumulated five amino acid substitutions in CDR1 and CDR2 upon maturation against lysozyme. Three of these residues have hydrophobic side chains, replacing serines, and participate in the hydrophobic core of the CDR1 in the mature antibody, suggesting that conformational rearrangements might occur in this loop during maturation. However, transition state analysis of the binding kinetics of mature cAb-Lys3 and germ line variants show that the maturation of this antibody relies on events late in the reaction pathway. This is reflected by a limited perturbation of k(a) and a significantly decreased k(d) upon maturation. In addition, binding reactions and the maturation event are predominantly enthalpically driven. Therefore, maturation proceeds through the increase of favorable binding interactions, or by the reduction of the enthalpic penalty for desolvation, as opposed to large entropic penalties associated with conformational changes and structural plasticity. Furthermore, the crystal structure of the mutant with a restored germ line CDR2 sequence illustrates that the matured hydrophobic core of CDR1 in cAb-Lys3 might be compensated in the germ line precursor by burying solvent molecules engaged in a stable hydrogen-bonding network with CDR1 and CDR2.

Amino Acid Sequence↗

PubMed2004

How thioredoxin can reduce a buried disulphide bond.

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.

Disulfides↗

PubMed2004

The structure of a triple mutant of pI258 arsenate reductase from Staphylococcus aureus and its 5-thio-2-nitrobenzoic acid adduct.

Structural insights into formation of the complex between the ubiquitous thiol-disulfide oxidoreductase thioredoxin and its oxidized substrate are under-documented owing to its entropical instability. In vitro, it is possible via a reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) to make a stable mixed-disulfide complex between thioredoxin from Staphylococcus aureus and one of its substrates, oxidized pI258 arsenate reductase (ArsC) from S. aureus. In the absence of the crystal structure of an ArsC-thioredoxin complex, the structures of two precursors of the complex, the ArsC triple mutant ArsC C10SC15AC82S and its 5-thio-2-nitrobenzoic acid (TNB) adduct, were determined. The ArsC triple mutant has a structure very similar to that of the reduced form of wild-type ArsC, with a folded redox helix and a buried catalytic Cys89. In the adduct form, the TNB molecule is buried in a hydrophobic pocket and the disulfide bridge between TNB and Cys89 is sterically inaccessible to thioredoxin. In order to form a mixed disulfide between ArsC and thioredoxin, a change in the orientation of the TNB-Cys89 disulfide in the structure is necessary.

Arsenite Transporting ATPases↗

PubMed2004

Crystallization of CcdB in complex with a GyrA fragment.

Plasmid addiction systems consist of a plasmid-encoded toxin-antidote pair that serves to stabilize low-copy-number plasmids in bacterial populations. CcdB, the toxin from the ccd system on the Escherichia coli F plasmid, acts as a gyrase poison. A 14 kDa fragment of gyrase, GyrA14, was found to bind to the toxin CcdB with an affinity of 1.75 x 10(-8) M. Crystals of the (GyrA14)(2) dimer in its free state belong to space group P4(3)2(1)2, with unit-cell parameters a = 86.4, c = 89.4 angstroms, and diffract to 2.4 angstroms. Crystals of the (GyrA14)(2)-(CcdB)(2) complex belong to space group P2(1)2(1)2(1), with a = 52.1, b = 83.3, c = 110.9 angstroms, and diffract to 2.8 angstroms resolution.

Bacterial Proteins↗

PubMed2004

Untangle, a tool for filtering overlapping diffraction patterns from multicrystals.

Standard crystallographic data-processing protocols are based on single-crystal models; data from aggregates of multiple crystals with different orientations are difficult to process. In certain cases, it is possible to separately index the diffraction patterns from the dominant crystals in the aggregate. Untangle is a program designed to identify and eliminate overlapping spots from such patterns in order to improve data quality. The program has a Python core with a simple and highly portable graphical user interface, permitting visual verification of the process and interactive modification of the overlap threshold. The software is available under an open-source license.

Bacterial Proteins↗

PubMed2004

Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin.

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.

Amino Acid Sequence↗

PubMed2003

Structural basis of carbohydrate recognition by the lectin LecB from Pseudomonas aeruginosa.

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.

Binding Sites↗