毒力岛基因SPI-1和SPI-2双缺失对肠炎沙门菌形态、毒力和免疫原性影响的研究  

Effect of double deletion of the pathogenicity islands SPI-1 and SPI-2 on the morphology,virulence,and immunogenicity of Salmonella enterica

作　　者：刘高松 吕雪莲 田秋丰 衣菲 于申业[1] LIU Gao-song;LV Xue-lian;TIAN Qiu-feng;YI Fei;YU shen-ye(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室/动物细菌病创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第6期553-560,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2022YFF0710500);国家自然科学基金(32172853、32373013);中央基本科研业务费(1610302022001)。

摘　　要：沙门菌毒力岛基因SPI-1和SPI-2分别编码Ⅲ型分泌系统(T3SS)中的T3SS-1和T3SS-2,T3SS可以将沙门菌分泌的效应蛋白转运至宿主细胞中。为分析毒力岛基因SPI-1和SPI-2缺失对肠炎沙门菌生物学特性、致病性和免疫原性的影响,本实验室前期构建了肠炎沙门菌SM6株毒力岛基因SPI-1和SPI-2双缺失株(SM6ΔT3SS),本研究通过绘制亲本株和缺失株的生长曲线,采用扫描电镜观察二者的形态,并测定二者对人结直肠腺癌细胞(Caco-2细胞)的黏附性与侵袭力。生长曲线结果显示,培养5 h时,SM6ΔT3SS株的生长速度显著快于亲本株SM6(P<0.05),自6 h起SM6ΔT3SS株的生长速度极显著快于SM6株(P<0.01)。扫描电镜观察可见,亲本株菌体多单个存在,而缺失株则出现明显的菌体聚集,且其表面被类似胶状物包裹。黏附性与侵袭力结果显示,与亲本株SM6相比,缺失株SM6ΔT3SS黏附及侵入Caco-2细胞的数量均极显著下降(P<0.001、P<0.001)。上述结果表明,毒力岛基因SPI-1和SPI-2的缺失可能改变了细菌的生长、代谢、分泌及或结构等特征,导致沙门菌的生物学特性的改变及降低了其对肠上皮细胞的黏附性和侵袭力。以1×10^(7)cfu/100μL的剂量经腹腔注射感染7日龄SPF鸡,并于感染后不同时间检测缺失株在SPF鸡盲肠、肝脏和脾脏的载菌量;将缺失株以1×10^(7)cfu/100μL的剂量经腹腔注射免疫7、21和35日龄鸡,在首免后不同时间采血,采用间接ELISA检测各组鸡血清中的IgG抗体水平;在首免后42 d时经腹腔注射1×10^(9)cfu/100μL/只的沙门菌SM6株,并于攻毒后7 d和14 d采血连同免疫期间采集的血一起采用间接ELISA检测各组鸡血清中IL-4和IFN-γ的分泌水平。在攻毒后14 d内观察并统计各组鸡的临床症状及死亡率。攻毒后7 d各组均剖杀部分鸡,采集其盲肠和肝脏组织制备病理切片观察各组织病理变化,评价该缺失株的免疫效力。载菌量结果显示,感染后2 d,缺�The Salmonella virulence island genes SPI-1 and SPI-2 encode T3SS-1 and T3SS-2 in the typeⅢsecretion system(T3SS),respectively,and which can transport effector proteins secreted by Salmonella into host cells.In order to analyze the effects of the deletion of virulence island genes SPI-1 and SPI-2 on the biological characteristics,pathogenicity and immunogenicity of Salmonella enterica,the laboratory constructed a double deletion strains of the virulence island genes SPI-1 and SPI-2 in the Salmonella enterica strain SM6(SM6ΔT3SS)in the previous period,and plotted the growth curves of the parental strain and the deletion strain.The morphology of the two cells was observed by scanning electron microscopy,and the adhesion and invasion of the parental strain SM6 and the deletion strain SM6ΔT3SS to human colorectal adenocarcinoma cells(Caco-2 cells)were determined.The growth curves showed that the growth rate of the SM6ΔT3SS strain was significantly faster than that of parental strain SM6 at 5 hours(P<0.05),and the growth rate of the SM6ΔT3SS strain was highly significant faster than that of the SM6 strain from 6 hours onwards(P<0.01).Scanning electron microscopic observation showed that the parental strains were mostly present singly,while the deletion strains had obvious bacterial aggregation,and their surfaces were coated by a gel-like substance.The results of adhesion and invasion showed that the deletion strain SM6ΔT3SS adhered to and invaded Caco-2 cells in a highly significant decrease compared with the parental strain SM6(P<0.001,P<0.001).These results suggest that the deletion of virulence island genes SPI-1 and SPI-2 may have altered the growth,metabolism,secretion and/or structure of bacteria,resulting in the alteration of the biological characteristics of Salmonella and the reduction of its adherence and invasion ability to intestinal epithelial cells.The 7-day-old SPF chickens were infected by intraperitoneal injection at the dose of 1×10^(7)cfu/100μL,and the bacteria-carrying capacity of the dele

关 键 词：肠炎沙门菌 Ⅲ型分泌系统 毒力岛 形态 毒力 免疫效力 减毒疫苗 

分 类 号：S852.61[农业科学—基础兽医学]