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

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,缺失株在鸡脾脏和肝脏中的载菌量极显著低于亲本株(P<0.001),但在盲肠中的载菌量极显著高于亲本株(P<0.01);感染后4 d,缺失株在肝脏和盲肠中的载菌量极显著和显著低于亲本株(P<0.01、P<0.05);感染后8 d,缺失株在脾脏中的载菌量极显著低于亲本株(P<0.01),且两组鸡在上述脏器中的载菌量均降至较低水平;抗体和细胞因子的ELISA检测结果显示,首免后14 d,免疫组鸡的抗体水平显著高于对照组(P<0.05)。从免疫后21 d,免疫组鸡的抗体水平均极显著高于对照组鸡(P<0.001);免疫期血清中IFN-γ的分泌水平无明显变化,IL-4的含量在免疫后42 d极显著高于对照组(P<0.01);攻毒后鸡血清中IFN-γ和IL-4的含量均显著高于对照组(P<0.05)。整个实验期SM6ΔT3SS免疫组鸡均存活且无明显的临床症状、无死亡,肝脏和肠黏膜仅出现轻微病理损伤,对照组鸡组鸡攻毒后14 d内死亡率达80%,肝脏和肠黏膜均出显较明显的病变。上述结果表明,SM6ΔT3SS株能够刺激SPF鸡产生较高水平的体液免疫和细胞免疫反应,对SPF鸡的致病性降低,并对鸡有较好的免疫保护效力。本研究为阐明沙门菌致病机制和研发安全有效的沙门菌减毒活疫苗奠定了良好基础,也为沙门菌病的防控提供了新思路。

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 deletion strain in the cecum,liver and spleen of the SPF chickens was detected at different times after infection;the chickens were immunized by intraperitoneal injection at a dose of 1×10^(7)cfu/100μL with the deletion strain at the age of 7,21 and 35 days,respectively.Blood samples was collected at different times after the first immunization;and the blood of each group of chickens was analyzed by indirect ELISA,and the blood of each group of chickens was analyzed by indirect ELISA.Blood was collected at different times after the first immunization and indirect ELISA was used to detect the IgG antibody levels in the serum of chickens in each group.The serum levels of IL-4 and IFN-γsecretion in each group were detected by indirect ELISA.The clinical symptoms of the chickens in each group were observed within 14 days after the first immunization,and the mortality rate of the chickens in each group was counted.At 7 days post-toxicity,some chickens in each group were dissected and their cecum and liver tissues were collected to observe the histopathological changes of the tissues and to evaluate the immunological efficacy of the deletion strain.The results of bacterial load showed that 2 days after infection,the bacterial load of the deletion strain in spleen and liver was extremely significantly lower than that of the parental strain(P<0.001),but the bacterial load in cecum was extremely significantly higher than that of the parent strain(P<0.01).On the 4 days after infection,the bacterial load of the deletion strain in liver and cecum was extremely significantly and significantly lower than that of the parent strain(P<0.01,P<0.05).On the 8 days after infection,the bacterial load of the deletion strain in spleen was extremely and significantly lower than that of the parent strain(P<0.01),and the bacterial load in the above organs of chickens in both groups dropped to a lower level.The ELISA results of antibodies and cytokines showed that the antibody level of chickens in the immunized group was significantly higher than that of the control group at 14 days after the first immunization(P<0.05).From 21 days after immunization,the antibody levels of all chickens in the immunized group were extremely significantly higher than those of the control group(P<0.001).There was no significant change in serum IFN-γsecretion level during the immunization period,but the content of IL-4 was significantly higher than that of the control group at 42 days after immunization(P<0.01).The content of IFN-γand IL-4 in the serum of SPF chickens was significantly higher than that of the control group after the tapping of the virus(P<0.05).The chickens in the SM6ΔT3SS-immunized group survived throughout the experimental period with no obvious clinical symptoms,no deaths,and only slight pathological damage to the liver and intestinal mucosa,while the chickens in the control group had a mortality rate of 80%within 14 days after the attack,with obvious lesions on the liver and intestinal mucosa.These results indicate that the SM6ΔT3SS strain can stimulate the production of high levels of humoral and cellular immune responses in SPF chickens,reduce the pathogenicity of SPF chickens,and have better immunoprotective effects on chickens.The construction of the double-deficiency strain laid a good foundation for elucidating the pathogenic mechanism of Salmonella and developing safe and effective live attenuated Salmonella vaccines,and also provides a new ideas for the prevention and control of Salmonellosis.