Tao Shuan, Fang Yewei, Zheng Lin, Zhang He, Xu Yao, Liang Wei
Department of Clinical Laboratory, The First Affiliated Hospital of Ningbo University, Ningbo, China.
Department of Medical Laboratory, Bengbu Medical College, Bengbu, China.
Virulence. 2025 Dec;16(1):2530665. doi: 10.1080/21505594.2025.2530665. Epub 2025 Jul 15.
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 . 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 ( < 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 ( > 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 ( < 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 ( > 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.
肠球菌是革兰氏阳性球菌，被认为是医院获得性感染的病原体之一。CRISPR-Cas是一种具有针对外来入侵核酸的靶向防御功能的适应性免疫系统，在抗生素耐药性中起重要作用。在本研究中，我们旨在研究II-A型CRISPR-Cas介导的免疫以及粪肠球菌中耐药基因水平转移的分子机制。通过同源重组策略构建突变菌株。通过质粒转化效率证实了粪肠球菌CRISPR1/Cas系统对质粒转化的干扰。通过酶切和测序验证构建间隔序列S1和PAM区域重组质粒中的不同突变位置，以评估CRISPR编码免疫的影响。在野生型菌株中，含有间隔序列和PAM位点的质粒pAT28-S1-S9的转化效率降低（<0.05）。间隔序列区域第25和28位的单碱基突变消除了野生型菌株阻止含有间隔序列和PAM位点的质粒转化的能力（>0.05），而间隔序列第2、10、18、23位的单个错配不影响CRISPR-Cas系统阳性菌株干扰质粒转化的能力（<0.05）。在没有PAM的pS1-pΔPAM质粒和含有单突变的质粒的转化效率方面，野生型菌株和突变菌株之间没有显著差异（>0.05）。总之，CRISPR-Cas系统可以阻断匹配间隔序列的转化，间隔序列相邻基序（PAM）附近或内部的突变使质粒能够逃避CRISPR编码的免疫。
