Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark.
Int J Mol Sci. 2023 Jun 24;24(13):10585. doi: 10.3390/ijms241310585.
Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an gene as a single target to inhibit plasmid-mediated horizontal gene transfer in by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the gene. The transcriptional levels of the gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the gene at a rate of 66.4%. The repressed expression of the gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (, , and ) and increased the global regulator genes (, and ). The repressed gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.
细菌接合构成了人类病原体中抗生素耐药基因 (ARG) 传播的主要水平基因转移机制。通过干扰接合过程，可以阻止或减少 ARG 的传播。在这项研究中，我们探索了使用 基因作为单一靶标，通过 CRISPR 干扰 (CRISPRi) 系统抑制质粒介导的水平基因转移的可能性。设计了三个单导向 RNA (sgRNA) 来靶向 基因。测试了 CRISPRi 菌株中 基因的转录水平、与接合相关的基因和接合效率。我们进一步探讨了抑制 基因表达对群体感应 (QS) 系统和生物膜形成的影响。结果表明，构建的 CRISPRi 系统有效地抑制了 基因的转录水平，抑制率为 66.4%。 基因的抑制表达导致 RP4 质粒的接合率降低了 88.7%，显著抑制了与接合相关的基因（ 、 、 ）的表达，并增加了全局调控基因（ 、 、 ）的表达。抑制 基因的表达减少了自动诱导物 2 信号 (AI-2) 的消耗，降低了生物膜形成率，达到了 68.2%。这项研究的结果表明， 基因可以作为抑制接合的通用靶标。构建的可接合性 CRISPRi 系统有可能在 ARG 高风险地区使用。
