尼罗罗非鱼无乳链球菌荚膜多糖合成基因的表达及其与r荚膜唾液酸含量、菌株致病性的关系

摘要

为了解尼罗罗非鱼无乳链球菌(GBS)荚膜多糖合成基因的表达及其与荚膜唾液酸含量、菌株致病性的关系,本实验克隆了尼罗罗非鱼荚膜多糖合成基因cpsE、cpsK和neuA,通过qRT-PCR方法分析了这3个基因在不同培养温度下表达水平的变化,同时通过比色法测定了不同培养温度下GBS荚膜唾液酸含量的变化,通过人工感染实验分析了不同水温条件下GBS对罗非鱼的致病性.结果显示,cpsE、cpsK和neuA编码的氨基酸序列均具有保守的与荚膜多糖合成相关的酶活性位点,CpsE、NeuA与已知鱼源GBS(Ⅰa和Ⅰb型)和人源GBS(Ⅰa、Ⅰb和Ⅱ～Ⅸ型)相应序列的同源性均达到97％以上,而CpsK与鱼源、人源的序列同源性则分别为56％～100％和27％～100％.在不同培养温度下GBS cpsK和neuA基因表达水平的变化与荚膜唾液酸含量的变化一致;在较高温度(28和34℃)下培养的GBS荚膜唾液酸含量及菌株攻毒后罗非鱼的死亡率均随温度的升高而递增,而在22℃下培养的GBS唾液酸含量最高,攻毒后罗非鱼的死亡率却最低.本研究结果表明,GBS CpsK和NeuA在GBS荚膜多糖的唾液酸化中起重要作用,较低温度下GBS荚膜唾液酸的高含量有助于其在宿主体内的潜伏,而较高水温条件下细菌的强致病性可能还与除荚膜唾液酸外的某些重要的毒力因子的表达有关.%In order to understand the expressions of capsular polysaccharide synthetic gene of Streptococcus agalactiae (GBS) isolated from Oreochromis niloticus (Nile tilapia) and their effects on capsular sialic acid con-tent and bacterial pathogenicity, capsular polysaccharide synthetic genecpsE,cpsK andneuA of GBS from O. niloticus were cloned in the study. The expression levels of these genes at different temperature were detected by qPCR. Capsular sialic acid content was detected by colorimetric method. The mortality rate of O. niloticusinfec-ted with GBS cultured at different temperature was analyzed by artificial challenge test. The results showed that amino acid sequence analysis encoded bycpsE,cpsK andneuA of GBS had conservative enzyme active sites which were essential for the synthesis of capsular polysaccharide.CpsE and NeuA of GBS isolated from O. nilotic-us shared high homology with those of GBS isolated from human and other fish species (>97％). The identities of cpsK of GBS isolated fromO. niloticus with those of other fish species (serotype Ⅰa and Ⅰb) and human (serotype Ⅰa, Ⅰb and Ⅱ～Ⅸ) were 56％–100％ and 27％–100％, respectively. The expression levels of cpsK and neuA of GBS cultivated at different temperature were consistent with that of capsular sialic acid content. Capsular sialic acid content and the mortality rate of tilapia infected with GBS cultivated at 28 and 34 ℃ were increased with the rise of temperature. The mortality rate of tilapia after artificial challenge at 22 ℃ was the lowest though the highest capsular sialic acid content was observed. The research suggested that the expression levels of CpsK and NeuA played an important role in the process of sialylation of capsular polysaccharide. Higher capsular sialic acid con-tent of GBS cultivated at low temperature may provide protection for it to stay in the host. However, the expres-sions of some important virulence factors besides capsular sialic acid might be necessary for a strong bacteria pathogenicity for GBS cultivated at higher temperature.