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  Director : Névine El Solh (nelsolh@pasteur.fr)


  abstract

 

Staphylococci are the etiological agents of severe bacterial infections whose prevalence is particularily high in infectious pathology. Antibiotic multiresistance has led to threapeutic difficulties concerning hospital strains. In 2002, the Unit had two major research themes i) a study of antibiotic resistance, which contributes to the optimisation of their use in therapy and the conception of new molecules, and ii) an analysis of the binding properties of staphylococcal cell wall autolysins to human matrix proteins in order to better understand the physio-pathology of staphylococcal infections on implanted material.



  report

cale

Several types of interaction between Fibronectin and the central domain of the autolysin AtlC of Staphylococcus capraeJ. Allignet, I. Old and N. EL Solh, in collaboration with P. England.

The staphylococcal Atl-type autolysins consist of two enzymatic domains separated by a central area which is made up of three imperfectly repeated polypeptide (R1-R2-R3) each of which includes two GW (glycine-tryptophan) motifs. The central (R1-R2-R3) areas of the autolysins Atl (S. aureus), AtlE (S. epidermidis) and AtlC (S. caprae), which were fused to a Histidine hexapeptide, bind to several matrix proteins: fibronectin, fibrinogen, vitronectin and bone sialoprotein. The BIAcore optical biosensor was used in order to analyse the kinetics of interaction with immobilized fibronectin. The constant of interaction for the S. caprae strain are similar to those published for a S. epidermidis strain. The affinity for fibronectin of the domain of AtlC containing the repeated domains (R1-R2-R3) is significant (kd = 2.48 nM) and similar to that of proteins produced by S. aureus and streptococcal strains (kd = 3.16 Nm). The binding kinetics suggest the existence of two types of interactions of variable affinity and multiple sites of interaction (» 10) which could be located in either or both ligands. The binding kinetics for fibronectin of R3 or R1-R2 fused to a Histidine hexapeptide were also analyzed. The constant of dissociation of His6-AtlCR3 with fibronectin (0.5 nM) was weaker than that of His6-AtlC R1-R2 or His6-AtlCR1-R2-R3 (2.5 nM). The two types of interaction that were detected for AtlCR1-R2-R3 were observed for AtlCR1-R2 but not for R3. The purification of separate AtlCR1 and AtlCR2 fused with His6 will be necessary to clarify the discordances observed and this is currently under study. The delimitation of the region of fibronectin that interacts with AtlCR1-R2-R3 was elucidated by a method based on the selection of phages expressing heptapeptides with a strong affinity for AtlCR1-R2-R3 (Phage Display). Screening of 46 such permitted the selection of several fibronectin peptides in order to evaluate, by ELISA, their ability to inhibit AtlC binding to fibronectin. The peptide WQPPRARI completely inhibited the interaction. This sequence is found in the C terminal region of the fibronectin and is one of five sequences responsible for heparin binding in that region.

Emergence in French hospitals, since 1998, S. aureus strains having an unusual genotype encoding streptogramin resistance. - J. Haroche, A. Morvan, M. Davi, Jeanine Allignet and N. El Solh.

These strains carry the genes vat(A) and vgb(A) which encode an acetyltransferase that inactivates streptogramin A and a lyase inactivating streptogramin B, respectively. In previous studies, we had shown that these two genes were regularly associated with vga(A) which encodes an ABC protein which confers resistance to streptogramin A. The three genes were detected in a fragment of 12.1 kb common to plasmids of variable sizes (26 to 45 kb) which results from the cointegration of two plasmids: a plasmid similar to pAMß1 which contains contiguous vat-vgb and whose replication gene is interrupted by the insertion sequence IS257, and a plasmid similar to pSX267 and pSK41 which contains vga(A) and a functional replication gene.

The analysis of the genotype of resistance to streptogramins of 62 strains of S. aureus isolated in 14 French hospitals from 1976 to 2000 allowed the detection of 25 strains carrying vat(A) and vgb(A). These last, which were distributed between 19 SmaI genotypes, had been isolated in eight hospitals, and for 22 of them, the date of isolation ranged between 1998 and 2000. These two contiguous genes were localised in HindIII restriction fragments of 9.5 or 8 kb (21 and 4 strains, respectively). The HindIII fragments of 9.5 kb contained the pAMß1 replication gene whose relative position to vat(A)-vga(A) (1.45 kb) is similar to that observed in the plasmids vat(A)-vgb(A)-vga(A), which suggests a structural conservation in this area of the plasmids. In conclusion, the strains of S. aureus vat(A)-vgb(A) emerged with an increased frequency since 1998 and in the majority of the cases, they resulted in a plasmid rearrangement and the subsequent dissemination of the modified plasmid in distinct clones.

Structural and functional analysis of the Vga(A) protein - O. Chesneau, N. El Solh, in collaboration with Jean-Michel Betton, Sylvie Dartevelle, Elie Dassa, Jean-Luc Guesdon, Muriel Delepierre.

PTR n°55 aims to characterize the role of a 522 aa protein with two ABC domains and no hydrophobic domain in the resistance to streptogramins. Subcellular localization, genetic studies and preliminary results in the biochemical analysis of the Vga(A) protein suggest that this protein may interact with the staphylococcal membrane via at least one partner not identified so far. Its implication in the putative efflux of the antibiotic out of the staphylococcal cells remains to be determined. Monoclonal antibodies are currently tested to detect Vga(A) specific epitopes that may distinguish Vga(A) from all other Vga-like proteins found by the sequencing projects of the microbial genomes, especially those of Gram-positive bacteria (see the database ABSCISSE at http://www.pasteur.fr/recherche/unites/pmtg/abc/database.html ).

Activity of the National Reference Centre (CNR) for Staphylococci

The expertise of the CNR was principally focused on the study of taxonomy, typing and bacterial resistance. In 2001, 545 strains were analysed. A preoccupying factor was the dissimination in several French Hospitals of epidemic clones of S. aureus resistant to ß-lactam antibiotics with a reduced sensitivity to glycopeptides.

Keywords: Staphylococci, adhesins, streptogramins, glycopeptides



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Gallais, Beatrice,bgallais@pasteur.fr El Solh, Névine, « Head of the Unit »,nelsolh@pasteur.fr

Chesneau, Olivier, Chargé de recherche,chesneau@pasteur.fr

Old, Iain, Assistant,igold@pasteur.fr

Haroche, Julien, « PhD student »,jharoche@pasteur.fr

Trad, Salim, « PhD student »,strad@pasteur.fr

Allignet, Jeanine, Ingénieur position II,allignet@pasteur.fr

Davi, Marilyne, Technician 2D,mdavi@pasteur.fr

Morvan, Anne, Technician 2D,amorvan@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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