Characterization of ComC export and processing in Streptococcus sanguinis

描述（由申请人提供）：血链球菌是绿链球菌的一员，是牙齿表面牙菌斑生物膜最早的定植者之一。在这个生态位中，血链球菌被认为是通过与口腔中有害细菌的生长竞争而发挥有益作用的。然而，血链球菌也是感染性心内膜炎的主要原因，这是一种经常致命的心脏瓣膜感染。这项研究旨在确定有助于口腔和心脏环境健康的基因。在这方面，我们最近开始描述S. sanguinis com条例。这个调控最明显的功能是遗传能力——从环境中吸收自由的外源DNA的能力。根据以往对肺炎链球菌和变形链球菌的研究，我们假设com调控也可能对心内膜炎的毒力或生物膜的形成很重要。我们的初步结果表明，com调控不增强心内膜炎的毒力；然而，它似乎对生物膜的形成有显著的影响。这些发现表明，这一规律可能是口腔竞争力的重要决定因素。在肺炎链球菌系统的基础上，建立了血链球菌的能力调控模型。在肺炎链球菌中，能力诱导前体肽ComC被裂解为其活性形式，能力刺激肽（CSP），并分泌作为群体感应信号，诱导DNA摄取。通过具有肽酶活性的ABC输出蛋白ComA的活性，在一个保守的位点发生切割。我们的研究结果表明，血参ComC也作为CSP被加工和分泌。然而，在S. sanguinis ComC中缺乏保守的切割位点，并且没有发现ComC的同源蛋白或其他能够加工ComC的蛋白。尽管我们取得了进展，但尚不清楚血参调控诱导的许多特征。为了进一步表征这一重要系统的基本特征，我们提出了三个目标：(1)分离和确定分泌能力刺激肽（CSP）的序列；(2)通过筛选确定突变体的集合来鉴定血链球菌ComC的出口和加工系统；(3)研究血链球菌ComC在异源宿主中的出口和加工。总之，这些目标提供了一系列表征系统的方法，该系统可能以多种方式促进血链球菌在口腔内的竞争力。一旦该系统被更好地表征，我们的研究结果可能允许操纵com系统来增强血链球菌在口腔中的有利特性，而不会增强其对心内膜炎的毒力。

DESCRIPTION (provided by applicant): Streptococcus sanguinis is a member of the viridans streptococci and is among the earliest colonizers of the dental plaque biofilm on the tooth surface. Within this niche, S. sanguinis is believed to play a beneficial role by competing with the growth of harmful bacteria in the mouth. However, S. sanguinis is also a leading cause of infective endocarditis, an often lethal infection of the cardiac valves. This research aims to identify genes that contribute to fitness in both the oral and cardiac environments. In this regard, we have recently begun to characterize the S. sanguinis com regulon. The most apparent function of this regulon is genetic competence- the ability to incorporate free, exogenous DNA from the environment. Based on previous studies in Streptococcus pneumoniae and Streptococcus mutans, we hypothesized that the com regulon might also be important for endocarditis virulence or biofilm formation. Our preliminary results suggest that the com regulon does not enhance endocarditis virulence; however it appears to have a significant effect on biofilm formation. Such findings suggest that this regulon may be an important determinant of competitiveness in the mouth. A model of the competence regulon in S. sanguinis is being constructed based on the well characterized system of Streptococcus pneumoniae. In S. pneumoniae, the competence inducing precursor peptide, ComC, is cleaved to its active form, competence stimulating peptide (CSP), and secreted to serve as a quorum sensing signal, inducing DNA uptake. Cleavage occurs at a conserved site through the activity of ComA, an ABC exporter with peptidase activity. Our results suggest S. sanguinis ComC is also processed and secreted as CSP. However, the conserved cleavage site is lacking in S. sanguinis ComC and no ComA ortholog or other protein capable of processing ComC has been identified. Despite our progress, many features of S. sanguinis com regulon induction are not understood. To further characterize such fundamental aspects of this important system with S. sanguinis we propose three aims: (1) to isolate and determine the sequence of secreted competence stimulating peptide (CSP) (2) to identify the S. sanguinis ComC export and processing system by screening a collection of defined mutants (3) to examine export and processing of S. sanguinis ComC in a heterologous host. Together, these aims provide a range of approaches for characterization of a system that may contribute in multiple ways to the competitiveness of S. sanguinis within the mouth. Once this system is better characterized, our findings may allow manipulation of the com system to enhance favorable properties of S. sanguinis in the mouth without enhancing its virulence for endocarditis. Public Health Relevance: The bacterium Streptococcus sanguinis is an important component of dental plaque and may benefit people by competing against harmful bacteria in the mouth. We are characterizing a system in Streptococcus sanguinis that enables it to take up DNA from its surroundings and may also help it survive in the mouth. This study proposes to characterize the most fundamental components of this important system.