台灣魚隻分離之無乳鏈球菌溶血型表現與其基因分析

Abstract

無乳鏈球菌（Streptococcus agalactiae）為革蘭氏陽性球菌，又稱為B群鏈球菌（Group B Streptococcus，GBS），可感染吳郭魚、虹鱒、烏魚、雜交條紋鱸等魚類，於養殖期間造成嚴重的經濟損失。本研究於自98年至103年間，於養殖魚（吳郭魚、鱸魚等）分離之無乳鏈球菌中，鑑別出三種不同溶血型：α、β以及γ溶血型菌株。無乳鏈球菌的溶血性與其毒力相關，溶血素（hemolysin）由cyl操縱子（cyl operon）基因組成，而cyl操縱子主要由二元調控系統（two-component regulatory system，TCS）基因covS/R（control of virulence，sensor and regulator ）負責調控，covS/R亦可調控莢膜化（encapsulation）的程度。本研究之目的，在於分析各溶血型菌株表現性狀之差異並探討其溶血素基因序列之差異。在莢膜化及生長速率上，α及β溶血型菌株並無明顯差異，皆於18小時進入高原期（stationary phase）；在indian ink染色法觀察下，γ溶血型菌株莢膜化程度較高，生長速率亦較緩慢，於48小時進入高原期。而以PCR方式增幅cyl操縱子基因並比對其序列，發現α溶血型菌株在cylF基因含有1252 bp的嵌入序列（insertion sequence，IS）。此嵌入序列具有兩段開放讀序框架（open reading frame）負責編碼轉位酶（transposase），並於cylF基因起始碼（start codon）下游23bp處插入截斷cylF，以致於無法產生完整之CylF產物；β溶血型菌株之cyl基因組並無基因缺失；而γ溶血型菌株發現有整組cyl操縱子的缺失，並在其位置發現含有DNA重組酶（DNA recombinase）及中隔形成蛋白Maf（septum formation protein，Maf）等的14kb基因組島（genomic island）。三種溶血型之無乳鏈球菌株於covS/R調控基因上皆未發現變異。由本試驗研究結果得知，α溶血型菌株與β溶血型菌株的溶血素基因差異為cylF基因區域內的嵌入序列，而γ溶血型菌株則為cyl操縱子全段基因的缺失並被取代，而三種溶血型菌株莢膜表現有差異但covS/R調控基因則無變異。

Streptococcus agalactiae are Gram positive cocci, also known as Group B Streptococcus (GBS). S. agalactiae can infect numerous fish species, including tilapia, rainbow trout, mullet, and hybrid striped seabass, and cause economic losses during cultivation. From 2009 to 2014, it was found that all S. agalactiae strains isolated from cultured fishes, such as tilapia and seabass, had different hemolytic phenotypes including α, β and γ. GBS hemolysin is responsible for hemolysis, and it is encoded by cyl operon. cyl operon is mainly regulated by the two-component regulatory systems (TCS), covS/R (control of virulence, sensor and regulator). Besides hemolysis, covS/R can also regulate encapsulation. The objective of the present study was to compare phenotype differences among three hemolytic variant strains and investigate genetic composition of hemolysin genes. α and β hemolytic strains showed no difference on encapsulation and growth rate, and these two strains grew into stationary phase after 18 hours culture. γ hemolytic strain showed higher encapsulation by Indian ink staining and slower growth rate with the stationary phase of 48 hours. Genetic analysis of cyl operon among GBS strains by PCR and sequencing found a 1,252 bp insertion sequence (IS), located in cylF region of α hemolytic strains. The IS had two open reading frames, both encoding transposase. The IS interrupted cylF gene by insertion in 23 bp downstream of the ATG start codon of cylF region, causing incomplete mRNA transcription of cylF. β hemolytic strain showed no mutation in cyl operon. γ hemolytic strain lost whole cyl operon with replacement of 14 kb genomic island containing genes of DNA recombinase and septum formation protein, Maf, etc. No mutation of covS/R regulatory gene was found among all three hemolytic variant strains. The results of the present study indicated that the difference of hemolysin gene between α and β hemolytic strain was due to the IS inserted in cylF region, while in the case of γ hemolytic strain, whole cyl operon was deleted and replaced. Differences of encapsulation were found among three hemolytic strains, with no genetic difference of covS/R regulatory gene.