Determining the role of dialog between pathogenic streptococci in biofilm develop

描述（由申请人提供）：本项目旨在描述化脓性链球菌（A群链球菌，GAS）和无乳链球菌（B群链球菌，GBS）之间重叠的细菌群体感应（QS）系统，以及该系统在生物膜形成和毒力中的作用。GAS和GBS都是引起多种疾病的重要人类病原体，通常与生物膜有关，并导致显著的人类发病率和死亡率。了解它们之间的交流将有助于开发新的QS抑制剂来调节这两种生物的毒力。在GAS中，QS信号肽Shp2和Shp3促进生物膜的形成。这些Shp肽由两种Rgg调节剂Rgg2/Rgg3调节，而Rgg2/Rgg3又由Shp肽调节。在GBS中，一个与Shp2几乎相同的小疏水肽（shp）被发现直接邻近与Rgg2几乎相同的同源物，称为RovS。RovS已被证明是GBS中众多毒力基因的调节剂。我们的初步数据表明，GBS产生的Shp可以激活GAS中Shp2/3调控基因，这意味着GAS和GBS通过这个QS系统进行种间交流。观察到这些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，清楚地表明了解它们的作用机制的重要性。这些实验结果可能为QS抑制剂提供新的靶点，以防止生物膜的形成，降低与链球菌感染相关的发病率和死亡率。为了研究QS以及GAS和GBS之间的种间交流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Rgg2和RovS，以确定这些蛋白是否可以互补。由于Shp2/3已被证明能促进GAS中生物膜的形成，因此将测试每个突变体对生物膜发育的影响。为了确定肽表达如何与生物膜形成重叠，我们将使用RNASeq来确定浮游生长或生物膜中生长的GAS和GBS的转录组谱，以及是否存在Shp肽。最后，我们将在小鼠感染和携带模型中测试这些基因的缺失是否会改变GAS和GBS的毒力。综上所述，这些研究将深入了解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。来自突变研究的数据将使我们能够描述第一个允许不同种类链球菌之间交流的信号系统。

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.