Functional study of the Vga(A) protein implicated in resistance to the A component of streptograminsO. Chesneau, H. Ligeret, N. El Solh
Vga(A) is an ABC protein with two putative ATP binding domains but no transmembrane domain. Nevertheless, it is found associated with the membranes of staphylococcal cells carrying the resistance gene, and it is possible that the protein contributes to an increase of the elimination of the antibiotic by excretion across he membrane. We have genetically dissociated the two halves of the protein, and also replaced the aspartate residue at position 408. This residue lies within the Walker B region of the C-terminus of Vga(A). The presence of a lysine in place of aspartate 408 was associated with total loss of the resistance to SgA conferred by the intact native Vga(A). The crystal structure of the ABC protein HisP has been determined, and Asp408 in Vga(A) corresponds to Asp178 in HisP. Similar modification of the HisP monomer abolishes ATP binding and histidine transport by the protein in E. coli. Thus, our experiments indicate that ATP binding of Vga(A) is required for expression of the resistance to the A component of streptogramins in staphylococci.
Characterisation of a transposon, Tn5406, conferring resistance to streptogramin AJ. Haroche, J. Allignet, N. El Solh
A novel transposon, Tn5406 (5467 bp), was isolated from a clinical isolate of S. aureus. Tn5406 carries a variant of the vga(A) gene, encoding an ABC protein conferring resistance to streptogramin A, and also three genes with significant similarities to genes implicated in the transposition of Tn554. The sequences flanking the sequenced copy of Tn5406 were consistent with those of att554, the preferred insertion site of Tn554. Wild-type strains of S. aureus resistant to streptogramin A contain one to two copies of Tn5406, and the copies are found both on the chromosome and on plasmids.
Analysis of adhesive properties of staphylococcal autolysins J. Allignet, N. El Solh
We have isolated a gene encoding an autolysin from a clinical isolate of S. caprae itself isolated from a bone infection associated with a prosthesis. This autolysin, AltC is related to previously described staphylococcal autolysins: Alt (S. aureus), AltE (S. epidermidis), and Aas (S. saprophyticus). These proteins contain two enzymatic domains (N-acetylmuramoyl-L-alanine amidase and endo-ß-N-acetylglucosaminidase) separated by three imperfectly repeated peptide sequences each including two GW (glycine-tryptophan) motifs. Like Aas, AltC binds fibronectin. The central domains of AltC, Atl and AltE were purified by a process involving tagging with a histidine hexapeptide. All three fusion proteins bound fibronectin, evidence that this property is common to all staphylococcal Alt-type autolysins, independent of the species in which they are found. S. aureus strains also produce other fibronectin binding factors (Fn BPA and FnBPB) but no such factors could be found in other coagulase-negative staphylococci. Thus, it is likely that fibronectin-binding autolysins are particularly important in the pathogenicity of coagulase-negative staphylococci.
Analysis of SD (serine-aspartate) proteins in Staphylococcus capraeS. Aubert, J. Allignet, K. Dyke, N. El Solh
Two genes encoding SD proteins, sdrY and sdrZ, were isolated from a infectious strain of S. caprae and sequenced. SdrY (1051 aa) has a signal sequence (39 aa), an A domain (600 aa) with 35% identity with the aa sequence of the A domain of the S. aureus SD fibrinogen-binding protein (ClfB), a domain containing 154 SD dipeptides, and a cell wall anchor domain including the sequence LPDTG. The A domain of SdrY was purified and did not bind fibrinogen. SdrZ (539 aa) contains a signal sequence (30 aa), a domain of 59 aa (A), a region including 60 SD dipeptides, a domain of 210 aa (C) and a C-terminal hydrophobic sequence of 40 aa (H). The aa sequences of domains C and H are 58% identical to a similar region of the cell wall SD protein of S. epidermidis, SdrH. The domains A and C of SdrZ are being purified to allow analysis of their binding to matrix proteins.
Highlights of the activities of the National Reference Centre (NRC) for Staphylococci http://www.pasteur.fr/sante/clre/cadrecnr/staph/staph-activites.html N. El Solh, O. Chesneau, A. Morvan, S. Aubert, J. Allignet
The NRC developed a reference technique for detecting strains of S. aureus with diminished sensitivity to glycopeptides. The technique contributed to the detection of such strains in numerous French hospitals. Finding that such strains are present in hospitals has caused concerns among the members of the national "Veille Microbiologique" (microbiological surveillance), as it raises the possibility of therapeutic failure. Most of the strains isolated during the year 2000 were monoclonal and related to those isolated in previous years. Therefore, their incidence can be reduced by measures limiting the dissemination of strains (hygiene and patient isolation).
The ica operon involved in the production of biofilm may be a marker of pathogenicity, and allow discrimination between infectious strains of S. epidermidis causing bone infections associated with prostheses, and commensal strains of the skin flora which can contaminate samples. Indeed, the operon is significantly more prevalent among infectious (80.5%) than commensal (17.4%) strains