PDF(Pubmed)
Abstract：
Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides\' coordinated survival in the natural environment and the mechanisms that infect the host.
摘要：
志贺洛类假单胞菌,革兰氏阴性杆菌,是肠杆菌科的唯一成员，能够产生极性和外侧鞭毛并引起人类胃肠道和肠外疾病。志贺氏菌的鞭毛转录层次目前未知。在这项研究中,我们确认了FlaK,FlaM,Flia,FliAL是志贺氏菌中负责极性和侧向鞭毛调节的四种调节剂。为了确定志贺氏菌的鞭毛转录层次，WT和ΔflaK的转录组，ΔflaM,ΔFIA,在这项研究中，进行了ΔfliAL的比较。定量实时聚合酶链反应（qRT-PCR）和发光筛选试验用于验证RNA-seq结果，电泳迁移率变化分析(EMSA)结果表明,FlaK可以直接与fliK的启动子结合,FLIE,flha,chey,虽然FlaM蛋白可以直接与flgO的启动子结合，flgT,和flgA。同时,我们还观察到VI型分泌系统（T6SS）和II型分泌系统2（T2SS-2）基因在转录组谱中下调，杀伤试验显示对ΔflaK的杀伤能力较低，ΔflaM,ΔFIA,和ΔFLAL与WT相比，表明鞭毛等级系统和细菌分泌系统之间存在串扰。入侵试验还表明，ΔflaK，ΔflaM,ΔFIA,和ΔfliAL在感染Caco-2细胞方面不如WT有效。此外,我们还发现鞭毛调节因子的缺失导致志贺氏菌的一些生理代谢基因的差异表达。总的来说,这项研究旨在揭示控制志贺氏菌鞭毛基因表达的转录层次，以及运动性之间的串扰，毒力,以及生理和代谢活动，为将来研究志贺洛芝在自然环境中的协调生存和感染宿主的机制奠定基础。
