The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo.
The Yersinia enterocolitica Ysa type III secretion system is expressed during infections both in vitro and in vivo.
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DOI：
10.1002/mbo3.136
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发表时间：
2013-12
期刊：
影响因子：
3.4
通讯作者：
Young, Glenn M.
中科院分区：
文献类型：
--
作者：
Bent, Zachary W.;Branda, Steven S.;Young, Glenn M.
小肠结肠炎耶尔森菌生物变种1B维持两个参与发病的III型分泌系统（T3SS），质粒编码Ysc T3SS和染色体编码Ysa T3SS。体外实验表明，Ysa T3SS仅能在26°C的高营养培养基中表达，培养基中含有高浓度的盐，这是一种人工条件，无法清楚地了解小肠结肠炎菌在宿主体内对信号的反应性质。然而，先前的研究表明，Ysa系统在小鼠胃肠道组织的定植中起作用。本研究通过构建一系列与绿色荧光蛋白基因（gfp）融合的Ysa启动子来分析该T3SS在感染过程中的表达。采用报告菌株，体外用HeLa细胞进行感染，体内用耶尔森菌病小鼠模型进行感染。绿色荧光蛋白（GFP）在体外和体内通过yspP启动子（编码分泌效应蛋白）和orf6启动子（编码T3SS装置的结构成分）的表达进行检测。在HeLa细胞感染期间用荧光显微镜检测GFP强度，在小鼠感染期间用流式细胞术检测组织中GFP的表达。这些方法，结合定量逆转录聚合酶链反应（qRT-PCR）对yspP mRNA转录物的定量，证明了Ysa系统在体外以接触依赖的方式表达，并在小鼠感染期间在体内表达。
Yersinia enterocolitica biovar 1B maintains two type III secretion systems (T3SS) that are involved in pathogenesis, the plasmid encoded Ysc T3SS and the chromosomally encoded Ysa T3SS. In vitro, the Ysa T3SS has been shown to be expressed only at 26°C in a high-nutrient medium containing an exceptionally high concentration of salt – an artificial condition that provides no clear insight on the nature of signal that Y. enterocolitica responds to in a host. However, previous research has indicated that the Ysa system plays a role in the colonization of gastrointestinal tissues of mice. In this study, a series of Ysa promoter fusions to green fluorescent protein gene (gfp) were created to analyze the expression of this T3SS during infection. Using reporter strains, infections were carried out in vitro using HeLa cells and in vivo using the mouse model of yersiniosis. Expression of green fluorescent protein (GFP) was measured from the promoters of yspP (encoding a secreted effector protein) and orf6 (encoding a structural component of the T3SS apparatus) in vitro and in vivo. During the infection of HeLa cells GFP intensity was measured by fluorescence microscopy, while during murine infections GFP expression in tissues was measured by flow cytometry. These approaches, combined with quantification of yspP mRNA transcripts by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), demonstrate that the Ysa system is expressed in vitro in a contact-dependent manner, and is expressed in vivo during infection of mice.
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