Mechanism of β-lactam action in streptococcus pneumoniae: The piperacillin paradox

Abstract

We thank E. Breukink for the generous gift of lipid II, F. Couppey for initiating the work, and G. Cerardi for technical support in the construction of strain sspCM99. We thank R.-L. Revel-Goyet, F. Lacroix, and J.-P. Kleman (Institut de Biologie Structurale, Grenoble, France) for support and access to the microscopy platform for time-lapse acquisitions and C. Morisco and G. Schoehn for support and access to the electron microscopy platform. J. Philippe was supported by a grant from la Région Rhône-Alpes. The work of R. Hakenbeck and D. Denapaite was supported by a grant from the Deutsche Forschungsgemeinschaft, Ha 1011/11-1. This work was partly funded by the COOPOL Innovation France-Chinese program and by the ANR (grant ANR-2011-BSV5-012-01 NOBLEACH), and it used platforms of the Grenoble Instruct Centre (ISBG; grant UMS3518 CNRS-CEA-UJF-EMBL) with support from the FRISBI (grant ANR-10-INSB-05-02) and GRAL (grant ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The human pathogen Streptococcus pneumoniae has been treated for decades with β-lactam antibiotics. Its resistance is now widespread, mediated by the expression of mosaic variants of the target enzymes, the penicillin-binding proteins (PBPs). Understanding the mode of action of β-lactams, not only in molecular detail but also in their physiological consequences, will be crucial to improving these drugs and any counterresistances. In this work, we investigate the piperacillin paradox, by which this β-lactam selects primarily variants of PBP2b, whereas its most reactive target is PBP2x. These PBPs are both essential monofunctional transpeptidases involved in peptidoglycan assembly. PBP2x participates in septal synthesis, while PBP2b functions in peripheral elongation. The formation of the "lemon"-shaped cells induced by piperacillin treatment is consistent with the inhibition of PBP2x. Following the examination of treated and untreated cells by electron microscopy, the localization of the PBPs by epifluorescence microscopy, and the determination of the inhibition time course of the different PBPs, we propose a model of peptidoglycan assembly that accounts for the piperacillin paradox.