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  Director : COURVALIN Patrice (pcourval@pasteur.fr)



The Antibacterial Agents Unit studies the genetic support, biochemical mechanisms, heterospecific expression, evolution, and dissemination of antibiotic resistance in bacterial pathogens for humans; in particular: enterococci and glycopeptides, Gram-negative bacilla and aminoglycosides. It has also developped trans-kingdom gene transfer from bacteria to mammalian cells.



A mutation in 23S rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae

Macrolides are largely prescribed for empiric therapy of community acquired respiratory tract infections and may be useful in case of intolerance or resistance to ß-lactams. The most prevalent mechanisms of macrolide resistance in S. pneumoniae are mediated by the ermB gene which encodes a 23S rRNA methylase that dimethylates A2058, resulting in macrolide-lincosamide-streptogramins B resistance (MLSB) and mef(A), the structural gene for an efflux pump specific for 14- and 15-membered macrolides. Recently, clinical isolate S. pneumoniae BM4455 was found to be resistant to 16-membered macrolides and to streptogramins. This unusual resistance phenotype was due to an A2062C (Escherichia coli numbering) mutation in domain V of the four copies of 23S rRNA. With the emergence of new resistance mechanisms, it seems advisable to test in vitro the activity of one member each of 14-, 15-, and 16-membered macrolide, ketolide, lincosamide and streptogramin class of drugs


Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii

Multidrug-resistant strain A. baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encode proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative two-component regulatory system might be involved in the regulation of expression of the adeABC gene cluster.


Regulation of expression of the vanD glycopeptide resistance gene cluster in Enterococcus

A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in E. faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanRD-VanSD two-component regulatory system were cotranscribed from the PRD promoter, whereas transcription of the vanYD, vanHD, vanD, vanXD, and intD genes was initiated from the PYD promoter located between vanSD and vanYD. The VanRD-VanSD regulatory system is likely to activate transcription of the resistance genes from promoter PYD. Glycopeptide-susceptible derivatives of BM4339 were obtained by trans-complementation of the frameshift mutation in the ddl gene, restoring a functional D-alanine:D-alanine ligase activity in this strain. Glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanYD D,D-carboxypeptidase, the VanHD dehydrogenase, the VanD ligase, the VanXD D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanRD-VanSD, apparently dependent, transcription from promoter PYD correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanXD gene was transcribed in BM4339 but detectable amounts of VanXD D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity only detectable under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus

Glycopeptide-dependence for growth in enterococci results from mutations in the ddl gene, which inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac, instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent E. faecium clinical isolates were determined. Each had a mutation consisting either in (1) a 5-bp insertion at position 41 leading to an early stop codon, (2) an in frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or (3) single base-pair changes resulting in an amino acid substitution (E13 ®  G, G99 ®  R, V241 ®  D, D295 ®  G, P313 ®  L). The consequences of these mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the E. coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

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  Office staff Researchers Scientific trainees Other personnel

GARNERO Sylvie, sgarnero@pasteur.fr

Van STEENKISTE Pascale, pvanstee@pasteur.rr

GALIMAND Marc, IP, Researcher, galimand@pasteur.fr

GUILLEMOT Didier, IP, Researcher, guillemo@pasteur.fr

MARCHAND Isabelle, IP, Postdoc, imarchan@pasteur.fr

ABADIA PATIÑO Lorena, Ph.D. Student, labadia@pasteur.fr

ADLEY Catherine, Ph.D., Invited Professor

ALONSO Rodrigo, Ph.D., Post-doctoral fellow, ralonso@pasteur.fr

BLANCHARD John, Ph.D., Invited Professor

BONORA Maria-Grazia, Ph.D. Student, bonoramg@pasteur.fr

CAO Thi Bao Van, Ph.D. Student

COLLOMBET Jean-Marc, Ph.D., post-doctoral fellow

DAHL Kristin, Ph.D.., post-doctoral fellow, krisdhal@pasteur.fr

GODREUIL Sylvain, M.D., Ph.D. student

GRILLOT-COURVALIN Catherine, M.D., Ph.D., Associate Prof. CNRS, ccourval@pasteur.fr

LAMBERT Thierry, Pharm. D., Ph.D., Associate Prof. Univers, tlambert@pasteur.fr

LEMANISSIER Véronique, Int. Pharmacy

MAGNET Sophie, Ph.D. Student

SABTCHEVA Stefana, M.D., Invited Professor

SUNDSFJORD Arnfinn, M.D., Invited Professor

CHAUVEL Murielle, Technician, IP, mchauvel@pasteur.fr

DEPARDIEU Florence, Technician, IP, fdepard@pasteur.fr

GERBAUD Guy, Engineer, IP, gerbaudg@pasteur.fr

GOUSSARD Sylvie, Technician, IP, sgouss@pasteur.fr

PERICHON Bruno, Engineer, IP, brunoper@pasteur.fr

SIMON Sylvie, Technician, INSERM, sysimon@pasteur.fr


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