Determining the role of dialog between pathogenic streptococci in biofilm develop

描述（由申请方提供）：本项目旨在表征化脓链球菌（A组链球菌，GAS）和无乳链球菌（B组链球菌，GBS）之间的重叠细菌群体感应（QS）系统以及该系统在生物膜形成和毒力中的作用。GAS和GBS都是重要的人类病原体，引起各种疾病，通常与生物膜相关，并导致显著的人类发病率和死亡率。了解它们之间的通信将允许开发新的QS抑制剂来调节两种生物体的毒力。 在GAS中，QS信号肽Shp 2和Shp 3增强生物膜形成。这些Shp肽由两个Rgg调节子Rgg 2/Rgg 3调节，Rgg 2/Rgg 3又由Shp肽调节。在GBS中，已经发现与Shp 2几乎相同的小疏水肽（shp）直接邻近与Rgg 2几乎相同的同系物，称为RovS。RovS已被证明是GBS中多种毒力基因的调节因子。我们的初步数据表明，生产的Shp GBS可以激活Shp 2/3调控基因的GAS标志GAS和GBS通过这个QS系统之间的种间通讯。这些QS系统在生物膜形成和毒力中的作用以及它们介导跨物种信号传导的能力清楚地表明了理解它们的作用机制的重要性。这些实验的结果可能为QS抑制剂提供新的靶点，以防止生物膜形成并降低与链球菌感染相关的发病率和死亡率。 为了检查QS和GAS与GBS之间的种间通信对毒力和生物膜形成的作用，我们将创建不含RovS和/或Shp肽的GBS菌株，并使用荧光素酶报告基因和qRT-PCR来测量敲除菌株中的基因表达。我们还将制作基因替代菌株，在GAS和GBS之间交换Rgg 2和RovS，以确定这些蛋白质是否可以相互补充。由于Shp 2/3已显示增强GAS中的生物膜形成，因此将测试每种突变体在生物膜发育中的作用。为了确定肽表达如何与生物膜形成重叠，我们将使用RNASeq来确定在存在或不存在Shp肽的情况下，促生长或在生物膜中生长的GAS和GBS的转录组谱。最后，我们将测试这些基因的缺失是否会改变GAS和GBS在感染和携带小鼠模型中的毒力。 总之，这些研究将为两种重要的人类病原体在临床相关环境中相互交流的能力提供很好的见解。来自突变研究的数据将使我们能够描述第一个允许不同链球菌物种之间通信的信号系统。

DESCRIPTION (provided by applicant): This project aims to characterize an overlapping bacterial quorum sensing (QS) system between Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) and the role of this system in biofilm formation and virulence. Both GAS and GBS are important human pathogens causing a variety of diseases, often biofilm related, and leading to significant human morbidity and mortality. Understanding communication between them will allow development of novel QS inhibitors to modulate virulence of both organisms. In GAS, the QS signaling peptides Shp2 and Shp3 enhance biofilm formation. These Shp peptides are regulated by two Rgg regulators, Rgg2/Rgg3 which, in turn, are regulated by the Shp peptides. In GBS, a small hydrophobic peptide (shp) almost identical to Shp2 has been found directly adjacent to a homolog almost identical to Rgg2 termed RovS. RovS has been shown to be a regulator of a multitude of virulence genes in GBS. Our preliminary data demonstrate that the production of the Shp by GBS can activate Shp2/3 regulated genes in GAS signifying interspecies communication between GAS and GBS via this QS system. The observed role of these QS systems in biofilm formation and virulence as well as their ability to mediate cross-species signaling clearly demonstrates the importance of understanding their mechanism of action. The results of these experiments may provide novel targets for QS inhibitors to prevent biofilm formation and decrease morbidity and mortality associated with streptococci infections. To examine the role of QS and interspecies communication between GAS and GBS on virulence and biofilm formation, we will create strains of GBS without RovS and/or the Shp peptide and use both luciferase reporters and qRT-PCR to measure gene expression in knockout strains. We will also make gene replacement strains swapping Rgg2 and RovS between GAS and GBS to determine whether these proteins can complement each other. Because Shp2/3 have been shown to enhance biofilm formation in GAS, the effects that each mutant has in biofilm development will be tested. To determine how peptide expression overlaps with biofilm formation, we will use RNASeq to determine the transcriptome profiles of GAS and GBS growing planktonically or in biofilms, and with or without the presence of the Shp peptide. Finally, we will test whether deletion of these genes alters the virulence of both GAS and GBS in mouse models of infection and carriage. Taken together, these studies will provide great insight into the ability of two important human pathogens to communicate with each other in clinically relevant settings. The data from mutational studies will allow us to characterize the first signaling system that allows communication between different species of streptococci.