Improving the lethal effect of Cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny.Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from-14.93 to-3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules. Copyright © 2013, American Society for Microbiology. All Rights Reserved.
This research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) to P. García (SAF2009-10824) and E. García (SAF2012-39444-C02-01) and from the MICINN (BFU2009-10052 and BF42012-36825) and the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Roberto Díez-Martínez was the recipient of a fellowship from the MICINN (FPI program).We declare a competing financial interest. We are coinventors on a Spanish patent application (no. P201330777) covering the results contained in this article. Any potential income generated by exploitation of the patent rights and received by our employers, the CSIC, and CIBERES shall be shared with us according to Spanish law