Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China.
Key Laboratory of Clinical Pharmacology of Antibiotics, National Heath Commission of the People's Republic of China, Shanghai, China.
Antimicrob Agents Chemother. 2023 Apr 18;67(4):e0154722. doi: 10.1128/aac.01547-22. Epub 2023 Mar 28.
Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted plasmid was increased from 3.96 × 10 to 2.01 × 10 compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of in ST15 by repressing CRISPR-Cas activity.
序列类型 (ST) 15 已成为耐碳青霉烯类肺炎克雷伯菌的新兴克隆，其中通常存在 I 型-ECRISPR-Cas，表明 CRISPR-Cas 系统可能无法阻止质粒的转移。本研究旨在探讨肺炎克雷伯菌 ST15 中质粒传播的机制。612 株非重复肺炎克雷伯菌 ST15 菌株（88 株临床分离株和 524 株来自 NCBI 数据库）中，98.0%存在 I 型-ECRISPR-Cas 系统。对 12 株 ST15 临床分离株进行全基因组测序，在 11 株分离株的质粒上发现了侧翼有原间隔区相邻基序（PAM）为 AAT 的自我靶向原间隔区。从一株临床分离株中克隆了 I 型-ECRISPR-Cas 系统，并在大肠杆菌 BL21(DE3)中表达。在含有 CRISPR 系统的 BL21(DE3)中，与空载体相比，带有 AAT PAM 的原间隔区携带质粒的转化效率降低了 96.2%，表明 I 型-ECRISPR-Cas 系统阻碍了质粒的转移。对已知抗 CRISPR（Acr）氨基酸序列的 BLAST 揭示了一种新型的 AcrIE9 样蛋白，与 AcrIE9 的序列同一性为 40.5%至 44.6%，命名为 AcrIE9.2，在携带 和 CRISPR-Cas 系统的 90.1%（146/162）ST15 菌株中均有发现。当 AcrIE9.2 在一株 ST15 临床分离株中克隆和表达时，与 AcrIE9.2 缺失株相比，CRISPR 靶向质粒的接合频率从 3.96×10 增加到 2.01×10。综上所述，AcrIE9.2 可能通过抑制 CRISPR-Cas 活性与 ST15 中质粒的传播有关。
