Membranes of the Dental Pathogen Streptococcus Mutans

 描述(申请人提供)：变形链球菌是一种普遍存在的口腔细菌，被鉴定为人类龋齿的主要病原体。某些变形链球菌也被鉴定为细菌性心内膜炎的病原体，在动脉粥样硬化斑块中被发现，并被证明入侵人类冠状动脉上皮细胞。仅与变形链球菌和其他致龋菌引起的龋齿相关的医疗保健费用就是巨大的，在美国每年超过十亿美元。本项目研究变形链球菌的膜生物发生和蛋白质分泌。由半透性脂质双层组成的膜包裹着所有细胞，通过决定什么可以通过，什么不可以通过嵌入其中的蛋白质，决定细胞的功能。变形链球菌具有细菌膜蛋白插入和蛋白分泌系统的共同特征，这些特征是研究最广泛的模式生物大肠杆菌以及线粒体和叶绿体的共同特征，但它也表现出多种独特的性质。由于这个原因，它正在赢得作为模式生物的声誉，以更好地了解其他链球菌和一般革兰氏阳性细菌中的蛋白质转运机制。所有已知的变形链球菌的毒力特性都是其膜蛋白组成和/或分泌的胞外蛋白的结果。由于生物膜由~50%的蛋白质组成，这些蛋白质是已知和现有的药物靶点，更好地了解变形链球菌的膜插入和运输途径，将其毒力因子引导到必要的位置，将有助于未来针对这种病原体和相关病原体的靶向治疗。本项目重点研究了两条关键的共翻译易位途径，即在所有活细胞中发现的信号识别颗粒(SRP)途径和在细菌/线粒体/叶绿体中发现的YidC插入酶途径。在革兰氏阳性菌中发现了另外一种称为YlxM的SRP成分，并在变形链球菌中发现了功能不同的双重YidC同源基因。变形链球菌特有的其他成分似乎也存在。在这个项目中，结合膜蛋白质组学、定向遗传、生化和生物物理方法，以及体内和体外的互补分析，将提高对变形链球菌膜蛋白插入和分泌途径的理解，并将识别与感受能力发育和突变体产生的关键毒力特性相关的特定的膜定位底物，因为分泌途径突变体在这方面存在缺陷。这些研究是通过建立方法学来促进的，以纯化变形链球菌的过渡活性核糖体，评估体内和体外的膜蛋白插入，并利用生物层干涉计量学、免疫共沉淀和化学交联法来检测和测量膜定位的分泌途径组件的相互作用，这些组件对本研究中确定的底物的运输和插入至关重要。

 DESCRIPTION (provided by applicant): Streptococccus mutans is a ubiquitous oral bacterium identified as a prominent etiologic agent of human dental caries. Certain strains of S. mutans have also been identified as causative agents of bacterial endocarditis, discovered within atherosclerotic plaque, and demonstrated to invade human coronary epithelial cells. The health care cost associated with dental decay stemming from S. mutans and other cariogenic organisms alone is enormous, over $64 billion annually in the USA. This project addresses membrane biogenesis and protein secretion in S. mutans. Membranes comprised of a semi-permeable lipid bilayer enclose all cells and, by dictating what can and cannot cross by virtue of proteins embedded within them, determine cellular function. S. mutans shares features common to bacterial membrane protein insertion and protein secretion systems common to the most widely studied model organism Escherichia coli, as well as to those of mitochondria and chloroplasts, but it exhibits multiple unique properties as well. For this reason it is gaining in reputation as a model organism to better understand the protein translocation machinery in other streptococci, and Gram-positive bacteria in general. All known virulence properties of S. mutans are a consequence of its membrane protein composition and/or secreted extracellular proteins. Since biological membranes are composed of ~50% proteins by mass, and these represent most known and existing drug targets, a better understanding of the membrane insertion and transport pathways of S. mutans that guide its virulence factors to their necessary locations will facilitate future targeted therapies against this and related pathogens. This projec focuses on two critical co-translational translocation pathways, the signal recognition particle (SRP) pathway found in all living cells, and the YidC insertase pathway found in bacteria/mitochondria/chloroplasts. The presence in Gram-positive bacteria of an additional SRP component called YlxM, and of dual YidC paralogs of differing function, was discovered in S. mutans. Additional components unique to S. mutans appear to exist as well. In this project, a combination of membrane proteomics, directed genetic, biochemical, and biophysical approaches, and complementary in vivo and in vitro analyses will improve the understanding of membrane protein insertion and secretion pathways of S. mutans and will identify specific membrane-localized substrates related to the pivotal virulence properties of competence development and mutacin production since secretion pathway mutants are defective in this regard. These studies are facilitated by the establishment of methodologies to purify transitionally active ribosomes from S. mutans, to assess membrane protein insertion in vivo and in vitro, and to utilize biolayer interferometry, co-immunoprecipitation, and chemical cross-linking to detect and measure interactions of membrane-localized secretion pathway components that are crucial to the transport and insertion of the substrates identified as part of this research.