Mechanistic study of the immune defense function of the CRISPR1-Cas system in Enterococcus faecalis.

Enterococci are Gram-positive cocci that are considered to be one of the causative agents of hospital-acquired infections. CRISPR-Cas is an adaptive immune system with targeted defense functions against foreign invading nucleic acids and plays an important role in antibiotic resistance. In this study, we aimed to investigate II-A CRISPR-Cas-mediated immunity and the molecular mechanism underlying the horizontal transfer of drug resistance genes in Enterococcus faecalis. The mutant strains were constructed by the homologous recombination strategy. The interference of plasmid transformation by the Enterococcus faecalis CRISPR1/Cas system was confirmed through plasmid transformation efficiency. The different mutation positions in the protospacer sequence S1 and PAM region recombinant plasmids were constructed through enzyme digestion and sequencing verification to assess the impact of the CRISPR-encoded immunity. In the wild-type strain, the transformation efficiency of plasmids pAT28-S1-S9 containing protospacers and PAM sites decreased (p < 0.05). Single-base mutations at positions 25 and 28 of the protospacer region eliminated the ability of the wild-type strain to prevent plasmid transformation containing the protospacer and PAM sites (p > 0.05), whereas a single mismatch at protospacer positions 2,10,18,23 did not affect the ability of CRISPR-Cas system-positive strains to interfere with plasmid transformation (p < 0.05). There was no significant difference between the wild-type strain and the mutant strain in the transformation efficiency of the pS1-pΔPAM plasmid without PAM and plasmids containing single mutations (p > 0.05). In conclusion, the CRISPR-Cas system can block the transformation of matching protospacer sequences, and mutations near or within the protospacer adjacent motif (PAM) allow the plasmid to escape CRISPR-encoded immunity.