Exploring the Ecological Roles of Mutanic Acid and Mutanicyclin; Two Novel Small Molecules Produced by Streptococcus mutans

项目摘要 龋齿是全球最常见的慢性疾病，伴随着巨大的经济负担， 并且在预防上影响最不能获得牙科护理和负担得起治疗的人群。链球菌 变链菌（mutans，Smu）是牙菌斑的常见成分，也是龋病的主要病原体。SMU 通过在牙齿表面形成坚固的生物膜并产生有机酸来溶解 牙齿的釉质。要坚持在牙菌斑社区和引起疾病，斯姆必须能够 直接胜过浮游生物。我们的研究小组最近发现了小分子突变 酸（MTA）、变环素（MTC）A-C及其生物合成基因簇（BGC）在全球几个 分布的Smu菌株，包括B 04 Sm 5，其分离自患有严重幼儿龋齿的儿童。 B 04 Sm 5，特别是MTC A，对其他口腔细菌具有显著的抗菌作用。此外，B 04 Sm 5 产生比Smu范例菌株UA 159更酸性的局部环境，并且这种表型是 依赖于一个功能正常的BGC。本提案的总体目标是确定MTA的作用， Smu生态学和毒力中的MTCs。我们提出了一个跨学科的研究方法，有两个具体目标： 目标1：了解BGC及其产品MTA和MTC的功能。MTA和MTC生产 和调控将通过液相色谱质谱和RNA-seq分析表征， BGC基因的缺失突变体。接下来，MTA和MTCs的活性谱将利用 成像质谱法（IMS）的竞争测定点蚀B 04 Sm 5，或ΔmtaD突变株，它不 不产生MTA或MTCs，对抗其他口腔细菌。IMS还将用于确定其他小型 与物种间竞争有关的分子。目标2：确定MTA、MTC A-C和 Smu基因编码蛋白对口腔生物膜毒力和群落功能的影响纯化的MTA，MTCs， 以及产MTA/MTC的Smu菌株对口腔微生物生态学和生物膜毒力的影响 使用我们建立的体外口腔生物膜模型，具有超过100个分类群，和下游元OMICS 分析。还将监测通过产酸和生物膜形成的群落毒力变化。的 拟议的研究提供了一个独特的机会，以扩大我们的知识的作用，BGC在口腔 微生物组，这是严重缺乏，并特别阐明了一种新的毒力途径，在斯穆。本研究 将大大增加我们对龋齿发病机制的理解，这代表了巨大的全球性 医疗负担，并可能指导新疗法和预防措施的开发。

Project Summary Dental caries is the most common chronic disease, globally, is associated with a tremendous economic burden, and disproportionally affects populations least able to access dental care and afford treatment. Streptococcus mutans (Smu) is a common constituent of dental plaque, and the primary etiologic agent of dental caries. Smu causes disease by forming robust biofilms on the tooth surface and producing organic acids which dissolve the underlying tooth enamel. To persist in the dental plaque community and cause disease, Smu must be able to directly outcompete commensal organisms. Our research group recently identified the small molecules mutanic acid (MTA), mutanicyclins (MTC) A-C and their biosynthetic gene cluster (mta BGC) in several globally distributed Smu strains, including B04Sm5, which was isolated from a child with severe early childhood caries. B04Sm5, and MTC A specifically, had significant antimicrobial effects on other oral bacteria. In addition, B04Sm5 produced a more acidic local environment than the Smu paradigm strain, UA159, and this phenotype was dependent on a functional mta BGC. The overarching goal of this proposal is to determine the role of MTA and MTCs in Smu ecology and virulence. We propose an interdisciplinary research approach with two specific aims: Aim 1: Functional insights of the mta BGC and its products MTA and MTCs. MTA and MTC(s) production and regulation will be characterized by liquid chromatography mass spectrometry and RNA-seq analysis of deletion mutants of mta BGC genes. Next, the spectrum of activity of MTA and MTCs will be explored utilizing imaging mass spectrometry (IMS) of competition assays pitting B04Sm5, or the ΔmtaD mutant strain, which does not produce MTA or MTCs, against other oral bacteria. IMS will also be used to identify additional small molecules pertinent to the interspecies competitions. Aim 2: Determine the impacts of MTA, MTC A-C, and mta encoding Smu on oral biofilm virulence and community function. The effects of purified MTA, MTCs, as well as MTA/MTC producing strains of Smu, on oral microbial ecology and biofilm virulence will be explored using our established in vitro oral biofilm model, featuring over 100 taxa, and downstream meta-OMICS analyses. Changes in community virulence via acid production and biofilm formation will also be monitored. The proposed study provides a unique opportunity to expand our knowledge of the role of BGCs in the oral microbiome, which is severely lacking, and specifically elucidate a novel virulence pathway in Smu. This research will significantly increase our understanding of the pathogenesis of caries, which represents a tremendous global medical burden, and is likely to guide development of new therapeutics and preventative measures.