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J. Biol. Chem., Vol. 279, Issue 16, 16463-16470, April 16, 2004

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A PBP2x from a Clinical Isolate of Streptococcus pneumoniae Exhibits an Alternative Mechanism for Reduction of Susceptibility to -Lactam Antibiotics^*

Lucile Pernot, Laurent Chesnel¶, Audrey Le Gouellec¶, Jacques Croizé||, Thierry Vernet¶, Otto Dideberg, and Andréa Dessen**

From the Laboratoire de Cristallographie Macromoléculaire and the ^¶Laboratoire d'Ingénierie des Macromolécules, Institut de Biologie Structurale Jean-Pierre Ebel (CNRS/CEA/UJF), 41 rue Jules Horowitz, 38027 Grenoble, France and the ^||Laboratoire de Bactériologie, Centre Hospitalier Universitaire, 38400 Grenoble, France

The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward -lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward -lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand 3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.

Received for publication, December 10, 2003 , and in revised form, January 13, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ605072.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number CAE53270

The atomic coordinates and structure factors (code 1RP5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by a fellowship (to L. C.) and a grant, both from the Région Rhône Alpes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a grant from ARECA.

^** To whom correspondence should be addressed: Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale J.-P. Ebel 41, rue Jules Horowitz, 38027 Grenoble, France. Tel.: 33-4-38-78-95-90; Fax: 33-4-38-78-54-94; E-mail: dessen{at}ibs.fr.

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