Zhang Kai, Wang Pengyu, Li Shanshan, Xie Xiaolei, Wang Zhenyu, Li Yang, Jiao Xinan, Li Qiuchun
Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, China.
Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University, Yangzhou 225009, China.
Vet Microbiol. 2024 Dec;299:110301. doi: 10.1016/j.vetmic.2024.110301. Epub 2024 Nov 15.
Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) provide prokaryotes with adaptive immunity against invasion by plasmids or phages. In Salmonella, the type I-E CRISPR-Cas system is typically considered silent in immunity against foreign genetic elements. To elucidate the role of the CRISPR-Cas system, we chose Salmonella enterica serovar Pullorum S06004 as a model organism due to its four spacers and well-defined biological characteristics observed in previous studies. Western blot analysis revealed expression of Cas3 in S06004 cultured in vitro, but plasmid transformation assays demonstrated that both wild-type (WT) and S06004 strains overexpressing LeuO (a positive regulator of CRISPR-Cas) showed no immunity against the target plasmid. RNA-Seq analysis detected significant downregulation of the fim cluster, encoding type I fimbriae, and T3SS1-related genes in the cas cluster mutant compared to the WT. This downregulation was further confirmed in mutants of CR1 and individual cas genes by qRT-PCR. Consequently, mutants of CR1 and cas clusters exhibited decreased invasion of chicken hepatocellular carcinoma cells. The consistent regulation of T3SS1 genes by the CRISPR-Cas system in S. Pullorum, S. Enteritidis, and S. Typhimurium indicates a common role for the type I-E CRISPR-Cas system in promoting bacterial virulence. However, the specific molecular mechanisms underlying this regulation require further investigation.
成簇规律间隔短回文重复序列及其相关的Cas蛋白（CRISPR-Cas）为原核生物提供了针对质粒或噬菌体入侵的适应性免疫。在沙门氏菌中，I-E型CRISPR-Cas系统通常被认为在抵抗外来遗传元件的免疫中处于沉默状态。为了阐明CRISPR-Cas系统的作用，我们选择肠炎沙门氏菌鸡白痢亚种S06004作为模式生物，因为在先前的研究中观察到它有四个间隔序列且生物学特性明确。蛋白质免疫印迹分析显示，在体外培养的S06004中Cas3有表达，但质粒转化试验表明，野生型（WT）和过表达LeuO（CRISPR-Cas的一个正调控因子）的S06004菌株对目标质粒均无免疫性。RNA测序分析检测到，与WT相比，编码I型菌毛的fim簇和cas簇中与三型分泌系统1（T3SS1）相关的基因在cas簇突变体中显著下调。通过定量逆转录聚合酶链反应（qRT-PCR）在CR1和单个cas基因的突变体中进一步证实了这种下调。因此，CR1和cas簇的突变体对鸡肝癌细胞的侵袭能力下降。鸡白痢沙门氏菌、肠炎沙门氏菌和鼠伤寒沙门氏菌中CRISPR-Cas系统对T3SS1基因的一致调控表明，I-E型CRISPR-Cas系统在促进细菌毒力方面具有共同作用。然而，这种调控背后的具体分子机制需要进一步研究。
