Roe Andrew J, Yull Helen, Naylor Stuart W, Woodward Martin J, Smith David G E, Gally David L
Zoonotic and Animal Pathogens Research Laboratory, Medical Microbiology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, Scotland, UK.
Infect Immun. 2003 Oct;71(10):5900-9. doi: 10.1128/IAI.71.10.5900-5909.2003.
Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEE1-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEE1-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.
肠道细菌的III型分泌系统可使效应蛋白转运至宿主细胞内。产志贺毒素大肠杆菌O157菌株分泌的蛋白包括转运装置的组分EspA、EspB和EspD，以及诸如转位紧密素受体（Tir）和线粒体相关蛋白（Map）等“效应蛋白”。本研究调查了LEE4转位子蛋白的调控，尤其是EspA。当大肠杆菌O157:H7在M9基本培养基中培养时，在细菌细胞表面检测不到EspA丝，但在用25 mM HEPES修饰的基本必需培养基中培养时，只有一部分细菌群体表达EspA丝。在指数生长后期检测到EspA丝化细菌的比例最高，此后丝状物迅速从细菌细胞表面消失。我们之前的研究表明，人和牛的大肠杆菌O157:H7菌株在EspD分泌水平上存在显著差异。此处证明，表达EspA丝的细菌群体比例与EspD分泌水平相关。如使用β-半乳糖苷酶和绿色荧光蛋白（GFP）启动子融合所证明的，单个细菌表达EspA丝的能力在LEE1 - 4操纵子转录水平不受控制。当使用LEE1 - 4翻译融合时，所有细菌，无论是否表达EspA丝，均显示出同等水平的GFP表达。尽管如此，与分泌且因此丝化细菌比例高的群体（高分泌者）相比，非分泌细菌比例高的群体（低分泌者）中LEE4 - espADB mRNA更为丰富。本研究表明，虽然需要特定的环境条件来诱导LEE1 - 4表达，但在细菌表面产生EspA丝并发生效应蛋白分泌之前还存在进一步的检查点。大肠杆菌O157:H7转位子表达中的这一检查点由作用于LEE4 - espADB mRNA的转录后机制控制。EspA丝化的异质性可能源于控制这种转录后机制的调节因子的相变表达。
