Nuevos antibacterianos basados en enzimas líticas de la pared celular de Streptococcus mitis y Streptococcus pneumoniae

Zusammenfassung

Phage endolysins and bacterial autolysins represent a novel class of antimicrobials (known also as enzybiotics) against bacteria due to their unique ability to cleave rapidly the peptidoglycan in a generally species-specific manner. The Skl endolysin is encoded by the ΦSK137 bacteriophage of Streptococcus mitis, an opportunistic pathogen closely related to Streptococcus pneumoniae. It comprises an N-terminal catalytic module (CHAP amidase family) and a C-terminal choline-binding module (CBM), involved in cell wall-attachment, that shares 64 % of sequence with the CBM of LytA, the major pneumococcal autolysin, whose catalytic module belongs to the Amidase_2 family. The comparative study of both amidases using different biophysical and biochemical techniques here reported has revealed that Skl has less affinity for choline than LytA, requires much higher concentrations of choline to form dimers, and displays a poor bacteriolytic activity against S. pneumoniae under conditions where LytA efficiently killed this pathogen. These results were indicative that their distinct capacity as enzybiotics could result, apart from dissimilar intrinsic activities of the catalytic modules, from sequence differences in their CBMs. If this were the case, replacement of the CBM of Skl by that of LytA would improve the choline binding affinity, the dimerization capacity and the bacteriolytic activity of the resulting chimeric lysin (QSLA2) in comparison to Skl wild type. By contrary, the equivalent substitution in LytA (chimeric lysin QLAS1) should depress these capacities with respect to LytA. The QLAS1 and QSLA2 chimeras were built from the genes of the parental enzymes by swapping the regions encoding the respective modules while preserving, in both constructions, the linker connecting the catalytic module to the CBM in the parental enzyme. The chimeric lysins showed high expression levels, which allowed their structural and functional characterization. As expected, QSLA2 showed higher choline affinity and dimerization ability than Skl and its antimicrobial activity was significantly improved in comparison to both Skl and LytA. Indeed, QSLA2 efficiently killed at very low concentration all the strains of S. pneumoniae and S. mitis tested, including multiresistant clinical isolates non-susceptible to LytA hydrolytic activity. By contrary, QLAS1, that comprises the catalytic module of LytA and the CBM of Skl, showed no bacteriolytic activity against S. pneumoniae and its choline binding features approached those exhibited by the Skl endolysin, in agreement, again, with the initial hypothesis. Of note, the lytic activity of QLAS1 on purified cell walls is comparable to that of QSLA2. Also, QSLA2 is able to protect zebrafish embryos from S. pneumoniae infection, which makes it a promising antimicrobial agent to treat pneumococcal infections. Taken together, our results provide new clues on the role played by the CBM and the module combination in the antimicrobial potential of this family of lysins, that could be useful to improve or modulate their activities, as well as to design new enzybiotics.