Abstract
Endolysins comprise a novel class of selective antibacterials refractory to develop resistances. The Cpl-7 endolysin, encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of a catalytic module (CM) with muramidase activity and a cell wall-binding module (CWBM) made of three fully conserved CW_7 repeats essential for activity. Firstly identified in the Cpl-7 endolysin, CW_7 motifs are also present in a great variety of cell wall hydrolases encoded, among others, by human and live-stock pathogens. However, the nature of CW_7 receptors on the bacterial envelope remains unknown. In the present study, the structural stability of Cpl-7 and the target recognized by CW_7 repeats, relevant for exploitation of Cpl-7 as antimicrobial, have been analyzed, and transitions from the CM and the CWBM assigned, using circular dichroism and differential scanning calorimetry. Cpl-7 stability is maximum around 6.0–6.5, near the optimal pH for activity. Above pH 8.0 the CM becomes extremely unstable, probably due to deprotonation of the N-terminal amino-group, whereas the CWBM is rather insensitive to pH variation and its structural stabilization by GlcNAc-MurNAc-l-Ala-d-isoGln points to the cell wall muropeptide as the cell wall target recognized by the CW_7 repeats. Denaturation data also revealed that Cpl-7 is organized into two essentially independent folding units, which will facilitate the recombination of the CM and the CWBM with other catalytic domains and/or cell wall-binding motifs to yield new tailored chimeric lysins with higher bactericidal activities or new pathogen specificities.
Highlights
Bacteriophages and bacteria produce a variety of peptidoglycan-degrading enzymes, the cell wall hydrolases (CWHs) [1], either to lyse host cells or to re-model the cell wall during growth and division
Cpl-7, the endolysin encoded by the Streptococcus pneumoniae bacteriophage Cp-7, consists of an N-terminal catalytic module (CM) with muramidase activity fused to a Cterminal module containing 3 identical tandem repeats of 42 amino acid residues each [9]
This paper reports thermal denaturation studies of the Cpl-7 endolysin aimed to examine its structural stability under a wide range of pHs and to identify the receptor of the CW_7 motifs on the bacterial surface, using both circular dichroism (CD) and differential scanning calorimetry (DSC)
Summary
Bacteriophages and bacteria produce a variety of peptidoglycan-degrading enzymes, the cell wall hydrolases (CWHs) [1], either to lyse host cells (endolysins) or to re-model the cell wall during growth and division (endogenous CWHs) Because of their potent ability to digest the cell wall of Gram-positive bacteria when exogenously added, endolysins represent a novel class of antimicrobials. The CW_7 repeats are essential for the activity [10] and unique among the sequences of the CWHs encoded by the pneumococcus and its bacteriophages [11] They confer to the Cpl-7 endolysin the ability to degrade pneumococcal cell walls containing either choline or ethanolamine and to retain full activity in the presence of high choline concentrations. The CM and the cell wall-binding module (CWBM) form an elongated particle of
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