CAJ | 학술논문

目的研究鼠疫耶尔森菌pCD1质粒上编码的假定蛋白YpCD1.08、YpCD1.09、YpCD1.16与pCD1质粒编码的Ⅲ型分泌系统(T3SS)在功能上的相关性.方法采用λ-Red重组系统构建鼠疫耶尔森菌YpCD1.08、YpCD1.09、YpCD1.16基因的突变株,通过测定分别培养于26℃下有钙、37℃下无钙和有钙TMH培养基中的以上3种突变菌株及野生菌株的生长曲线,分析突变菌株的低钙响应情况；在26℃和37℃条件下分别培养鼠疫菌突变菌株,并提取总RNA,采用定量PCR分析3种基因的转录水平及其对温度的依赖性；用β-内酰胺酶报告分子的方法分析3种基因的突变对效应蛋白转运的影响.结果在26℃和37℃低钙培养条件下,突变菌株△YpCD1.08、△YpCD1.09、△YpCD1.16的生长曲线与野生型菌株一致,表明其低钙响应正常.YpCD1.08、YpCD1.09、YpCD1.16基因在37 ℃与26℃转录水平的比值分别为2.3±0.3、2.3±0.5、3.2±0.7,表明这3个基因在鼠疫菌中均有转录,且其转录水平受温度调控,与T3SS基因转录水平的温度依赖性一致.β-内酰胺酶报告分子实验结果表明,突变菌株△YpCD1.08在140 min时477与520 nm波长的发光强度的比值为2.5,而野生型菌株仅为1.8,表明△YpCD1.08菌株对YopE的转运能力要高于野生株,而其他两个突变株与野生株类似.结论 YpCD1.08、YpCD1.09、YpCD1.16基因有可能是T3SS的新成员,假定蛋白YpCD1.08可能参与效应蛋白YopE的转运及其调控.
목적 연구서역야이삼균pCD1질립상편마적가정단백YpCD1.08、YpCD1.09、YpCD1.16여pCD1질립편마적Ⅲ형분비계통(T3SS)재공능상적상관성.방법 채용λ-Red중조계통구건서역야이삼균YpCD1.08、YpCD1.09、YpCD1.16기인적돌변주,통과측정분별배양우26℃하유개、37℃하무개화유개TMH배양기중적이상3충돌변균주급야생균주적생장곡선,분석돌변균주적저개향응정황；재26℃화37℃조건하분별배양서역균돌변균주,병제취총RNA,채용정량PCR분석3충기인적전록수평급기대온도적의뢰성；용β-내선알매보고분자적방법분석3충기인적돌변대효응단백전운적영향.결과 재26℃화37℃저개배양조건하,돌변균주△YpCD1.08、△YpCD1.09、△YpCD1.16적생장곡선여야생형균주일치,표명기저개향응정상.YpCD1.08、YpCD1.09、YpCD1.16기인재37 ℃여26℃전록수평적비치분별위2.3±0.3、2.3±0.5、3.2±0.7,표명저3개기인재서역균중균유전록,차기전록수평수온도조공,여T3SS기인전록수평적온도의뢰성일치.β-내선알매보고분자실험결과표명,돌변균주△YpCD1.08재140 min시477여520 nm파장적발광강도적비치위2.5,이야생형균주부위1.8,표명△YpCD1.08균주대YopE적전운능력요고우야생주,이기타량개돌변주여야생주유사.결론 YpCD1.08、YpCD1.09、YpCD1.16기인유가능시T3SS적신성원,가정단백YpCD1.08가능삼여효응단백YopE적전운급기조공.
Objective To investigate the functional relations between the putative proteins YpCD1.08,YpCD1.09,YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type Ⅲ secretion system (T3SS).Methods Mutants of YpCD1.08,YpCD1.09,YpCD1.16 were constructed using λ-Red recombinant system.The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 ℃ and 37 ℃ were determined to analyze the low calcium response phenotype.The transcription levels of △YpCD1.08,△YpCD1.09,△YpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 ℃ and 37 ℃ and extraction of RNA.A β-1actamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.Results When grown in TMH medium without calcium at 26 ℃ and 37 ℃,the growth curve of the YpCD1.08,YpCD1.09,YpCD1.16 mutants were similar to that of the wild-type strain,indicating that the low calcium response of all the mutants were normal.The ratios of YpCD1.08,YpCD1.09,YpCD1.16 gene transcriptional level at 37 ℃ and 26 ℃ were 2.3 ± 0.3,2.3 ± 0.5 and 3.2 ± 0.7,respectively,indicating that these genes were transcribed in Yesinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of YersiniaT3SS genes.The β-lactamases reporter assays demonstrated that AYpCD1.08 could translocate much higher level of YopE into HeLa cells,since that the light intensity ratio of 477/520 nm at 140 min was 2.5,whereas it was 1.8 for the wild-type strain,and the values in ＞YpCD1.09 and ＞YpCD1.16 were similar to the wild-type strain.Conclusion YpCD1.08,YpCD1.09,YpCD1.16 gene are likely to be the new members of T3SS,and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.