potA基因缺失对猪链球菌2型生物学特性及致病性影响的研究

Biological characteristics and pathogenicity of a potA-deficient mutant of Streptococcus suis serotype 2

为了探究potA基因对猪链球菌2型(SS2)生物学特性和致病性的影响,本研究利用同源重组系统构建potA基因缺失突变株(ΔpotA),并经PCR和western blot鉴定。采用显微镜观察、生长曲线测定和平板计数等方法比较ΔpotA和野生SC-19菌株的细胞形态和生长速率。结果显示,相比于SC-19菌株,ΔpotA菌株的链长极显著增长(P<0.001),生长速率下降,表明potA基因的缺失影响了SS2的正常生长;将不同浓度的ΔpotA和SC-19菌株分别感染小鼠,比较小鼠的存活率,利用平板计数法检测小鼠血液、脑、肺和脾等组织中的载菌量。结果显示,感染SC-19菌株的小鼠存活率远高于感染ΔpotA菌株的小鼠;两组小鼠血液中除感染后1 d和3 d的载菌量存在极显著差异外(P<0.01),其他各时间点两组小鼠的载菌量均无显著差异(P>0.05),但两组小鼠脑、肺和脾等组织中的载菌量均无显著差异(P>0.05),表明potA基因的缺失导致SS2对小鼠的致病性增强,但与SS2在组织中的定植能力无关;通过ΔpotA和SC-19菌株对HEp-2细胞的粘附及侵入试验和qRT-PCR检测两种菌中的粘附及侵入相关基因(srtA、aupA、sao、fbps、gapdH、eno、sadP、ccpA和dltA)的转录水平,进一步探究potA基因的缺失导致SS2对小鼠致病性增强的机制。结果显示,和SC-19菌株相比,ΔpotA对HEp-2细胞的粘附及侵入数量均极显著升高(P<0.01),srtA、fbps和sadP等与粘附及侵入相关基因的转录水平均显著上调(Fold change>2,P<0.001),表明potA基因的缺失促进了SS2粘附及侵入相关基因的转录,从而增强了SS2对HEp-2细胞粘附及侵入的能力。本研究首次证实,ΔpotA菌株的致病能力增强与菌株的自身生长速率和组织定植能力无关,而是由于potA基因的缺失促进了SS2粘附及侵入相关基因的转录,增强了其对细胞的粘附及侵入的能力。本研究为进一步研究SS2 potA基因的功能及致病机制提供了数据参考。

To investigate the effect of potA gene deletion on the biological characteristics and pathogenicity of Streptococcus suis type 2(SS2),a potA gene deletion mutant(ΔpotA) was constructed by homologous recombinant system and identified by PCR and western blot.The morphology and growth rate of ΔpotA and SC-19 strains were compared by microscopic observation,growth curve and plate count.The results showed that compared with SC-19 strains,the chain length of ΔpotA strains increased significantly(P<0.01),and the growth rate decreased,suggesting that the deletion of potA gene affected the normal growth of SS2.The survival rate and the bacterium capacity in blood,brain,lung and spleen tissues of mice infected with ΔpotA and SC-19 strains were compared.The results showed that the survival number of mice infected with SC-19 strains were much higher than that infected with ΔpotA strains.At 1 st and 3 rd day,the bacterium capacity in the blood of the mice infected with SC-19 and ΔpotA strains were significantly differentce(P<0.01),but there were no significantly differences in the bacterium capacity at other time points(P>0.05) and in other tissues(brain,lung and spleen)(P>0.05).These results indicated that the deletion of potA gene enhanced the pathogenecity of SS2,but it was not related to the colonization ability of SS2 in tissues.To further explore the mechanism of pathogenicity enhancement of SS2 in mice caused by the deletion of potA gene,the adhesion and invasion assays of SC-19 and ΔpotA strains to HEp-2 cell were designed and the transcriptional levels of some adhesion and invasion related genes(srtA,aupA,sao,fbps,gapdH,eno,sadP,ccpA and dltA) in the two strains were detected by qRT-PCR.The results showed that compared with SC-19 strains,the numbers of adhesion and invasion of ΔpotA strains to HEp-2 cell increased significantly(P<0.01),the trans-criptional levels of srtA,fbps,sadP and other genes related to adhesion and invasion were significantly up-regulated(fold change>2,P<0.001),indicating that the deletion of potA gene promoted the transcription of adhesion and invasion related genes,thus enhancing adhesion and invasion ability of SS2 to HEp-2 cells.This research we confirmed for the first time that the pathogenicity increasing of ΔpotA strain is due to the deletion of potA gene promoting the expression of genes associated with adhesion and invasion of SS2 and enhancing the adhesion and invasion ability to cells,which is independent on the growth rate and tissue colonization ability of SS2.This study provided scientific data for further study on the function and pathogenic mechanism of potA gene in SS2.