Thermal stability of Cpl-7 endolysin from the streptococcus pneumoniae bacteriophage Cp-7; cell wall-targeting of its CW_7 motifs.
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.
Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, and Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain.
Noemí
N
Bustamante
Palma
P
Rico-Lastres
Ernesto
E
García
Pedro
P
García
Margarita
M
Menéndez
D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D017510:Protein Folding; D017108:Streptococcus Phages / Q000201:enzymology*; D013296:Streptococcus pneumoniae / Q000821:virology*; D013696:Temperature
D010450:Endopeptidases; C001143:endolysin