Hanson Brett R, Slepenkin Anatoly, Peterson Ellena M, Tan Ming
Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA.
Department of Pathology and Laboratory Medicine, University of California, Irvine, California, USA.
J Bacteriol. 2015 Oct;197(20):3238-44. doi: 10.1128/JB.00379-15. Epub 2015 Jul 27.
The Scc4 protein (CT663) of the pathogenic bacterium Chlamydia has been described as a type III secretion (T3S) chaperone as well as an inhibitor of RNA polymerase. To examine if these roles are connected, we first investigated physical interactions between Chlamydia trachomatis Scc4 and the T3S chaperone Scc1 and a T3S substrate, CopN. In a yeast 3-hybrid assay, Scc4, Scc1, and CopN were all required to detect an interaction, which suggests that these proteins form a trimolecular complex. We also detected interactions between any two of these three T3S proteins in a pulldown assay using only recombinant proteins. We next determined whether these interactions affected the function of Scc4 as an inhibitor of RNA transcription. Using Escherichia coli as a heterologous in vivo system, we demonstrated that expression of C. trachomatis Scc4 led to a drastic decrease in transcript levels for multiple genes. However, coexpression of Scc4 with Scc1, CopN, or both alleviated Scc4-mediated inhibition of transcription. Scc4 expression also severely impaired E. coli growth, but this growth defect was reversed by coexpression of Scc4 with Scc1, CopN, or both, suggesting that the inhibitory effect of Scc4 on transcription and growth can be antagonized by interactions between Scc4, Scc1, and CopN. These findings suggest that the dual functions of Scc4 may serve as a bridge to link T3S and the regulation of gene expression in Chlamydia.
This study investigates a novel mechanism for regulating gene expression in the pathogenic bacterium Chlamydia. The Chlamydia type III secretion (T3S) chaperone Scc4 has been shown to inhibit transcription by RNA polymerase. This study describes physical interactions between Scc4 and the T3S proteins Scc1 and CopN. Furthermore, Chlamydia Scc1 and CopN antagonized the inhibitory effects of Scc4 on transcription and growth in a heterologous Escherichia coli system. These results provide evidence that transcription in Chlamydia can be regulated by the T3S system through interactions between T3S proteins.
致病细菌衣原体的Scc4蛋白（CT663）被描述为一种III型分泌（T3S）伴侣蛋白以及RNA聚合酶的抑制剂。为了研究这些作用是否相关，我们首先研究了沙眼衣原体Scc4与T3S伴侣蛋白Scc1以及T3S底物CopN之间的物理相互作用。在酵母三杂交试验中，Scc4、Scc1和CopN都是检测相互作用所必需的，这表明这些蛋白形成了一个三分子复合物。我们还在仅使用重组蛋白的下拉试验中检测到了这三种T3S蛋白中任意两种之间的相互作用。接下来，我们确定这些相互作用是否影响Scc4作为RNA转录抑制剂的功能。使用大肠杆菌作为异源体内系统，我们证明沙眼衣原体Scc4的表达导致多个基因的转录水平急剧下降。然而，Scc4与Scc1、CopN或两者共表达可减轻Scc4介导的转录抑制。Scc4的表达也严重损害了大肠杆菌的生长，但Scc4与Scc1、CopN或两者共表达可逆转这种生长缺陷，这表明Scc4对转录和生长的抑制作用可被Scc4、Scc1和CopN之间的相互作用所拮抗。这些发现表明，Scc4的双重功能可能作为一个桥梁，将T3S与衣原体中的基因表达调控联系起来。
本研究调查了致病细菌衣原体中调节基因表达的一种新机制。衣原体III型分泌（T3S）伴侣蛋白Scc4已被证明可抑制RNA聚合酶的转录。本研究描述了Scc4与T3S蛋白Scc1和CopN之间的物理相互作用。此外，衣原体Scc1和CopN在异源大肠杆菌系统中拮抗了Scc4对转录和生长的抑制作用。这些结果提供了证据，表明衣原体中的转录可通过T3S蛋白之间的相互作用由T3S系统调节。
