cel-EIIB蛋白调控罗非鱼无乳链球菌强毒、弱毒株毒力相关基因的表达模式

罗非鱼无乳链球菌纤维二糖-磷酸转移酶系统（cel-PTS）的EIIB蛋白对强毒株毒力影响有限,但对弱毒株毒力似乎存在潜在的影响,但具体机制仍不清晰,有必要弄清该蛋白调控强、弱毒株的毒力相关基因表达模式。在前期研究中,通过同源重组技术,构建了无乳链球菌强毒株cel-EIIB基因缺失株,本研究通过类似的方法,获得弱毒株该基因的缺失株。用无乳链球菌强毒、弱毒株及它们的cel-EIIB缺失株分别感染斑马鱼,结果显示,cel-EIIB缺失后,导致弱毒株毒力明显返强,而强毒株毒力则轻微减弱。qPCR检测发现,cel-EIIB缺失可致cel-PTS系统的cel-EIIA、双组分信号转导系统（TCS）的DltR和CiaH以及毒力基因sodA、cpsD和cpsG在强、弱毒株中呈现相反的表达模式;此外,TCS系统的RgfC、DltS和CsrR以及毒力基因cspA和pavA在强毒突变株中表达未受影响,但在弱毒突变株中的表达却显著上调。研究揭示,EIIB蛋白可能通过调控上述毒力相关基因表达而负调控弱毒菌株的毒力。

Analysis of expression patterns of virulence-related genes regulated by the cel-EIIB protein in virulent and attenuated strains of Streptococcus agalactiae in tilapia

Streptococcus agalactiae is an important pathogen associated with humans, animals, and fishes. Recently, it has spread globally, resulting in great economic losses in tilapia industries. The phosphotransferase system (PTS) is regarded as the "nervous system" of bacteria. The effect of the EIIB protein of cellobiose-PTS (cel-PTS) on S. agalactiae virulent strain virulence is limited, but it could potentially effect that of the attenuated strain; however, the specific mechanism is still unclear. Therefore, it is necessary to clarify the expression patterns of virulence-related genes regulated by the EIIB protein. In the previous study, the cel-EIIB gene deletion of the S. agalactiae virulent strain was constructed by homologous recombination technology. In this study, the cel-EIIB gene deletion of the S. agalactiae attenuated strain was obtained using a similar method. Zebrafish were infected with a S. agalactiae virulent strain, attenuated strain, and their cel-EIIB gene deleted strains, respectively. The results showed that S. agalactiae attenuated strain virulence was significantly stronger after cel-EIIB gene deletion, whereas S. agalactiae virulent strain virulence was only slightly reduced. It was confirmed by PCR detection that the deletion of cel-EIIB could cause cel-EIIA of the cel-PTS system, DltR and CiaH of the two-component signal transduction system (TCS), and sodA, cpsD, and cpsG of virulence genes showed opposite expression patterns in virulent and attenuated strains. In addition, the expression of RgfC, DltS, and CsrR of TCS and cspA and pavA of virulence genes were not affected in the mutant of the virulent strain, but up-regulated significantly in the mutant of the attenuated strain. It is speculated that the increased virulence of the S. agalactiae attenuated strain was related to the up-regulation of the expression of these virulence-related genes. To summarize, the EIIB protein may negatively regulate the virulence of attenuated strains by regulating the expression of the above virulence-related genes.