Determinants of Streptococcus pyogenes fitness in the female primate genital tract: A genome-wide analysis

项目摘要 人类细菌病原体化脓性链球菌（A组链球菌，GAS）引起超过 全世界每年有7亿人感染。这项拟议中的研究试图在基因组上发现- 大规模的，GAS基因所需的，或有助于健身的女性生殖道，感染部位， 我们对此知之甚少。我们将使用血清型M28 GAS菌株，因为 这些生物体与女性生殖道疾病的关联是非随机的， 包括产后败血症（产褥热）和新生儿侵入性感染。病原体基因和 生殖道相互作用的两个阶段的基本分子发病过程将是 研究：（一）定殖和增殖和（二）持久性。这种基础科学知识的缺乏 严重限制了创造新的临床工具，如成功的GAS疫苗的能力。因此，研究 旨在解决这一关键的知识缺陷。我们将结合联合收割机最近的成功应用 转座子定向插入位点测序（TraDIS）技术，以我们18年的生产经验， 食蟹猴GAS感染模型的经验，目前的金标准实验 动物来研究这种人类病原体。我们将使用TraDIS进行体内全基因组筛选， 系统地鉴定定植和增殖所需的GAS基因，以及在生殖器中的持久性 雌性食蟹猴的生殖道。这些研究的目的是确定新的分子 GAS使用的机制，以提高在这个解剖生态位的健身。我们的研究结果可能最终 有助于制定新的翻译策略，以预防或治疗GAS感染。为了实现我们的目标， 提出了以下两个具体目标：具体目标1：利用TraDIS进行全基因组鉴定， GAS基因需要（i）定殖和增殖，和（ii）持久性（即，生殖道的问题 雌性食蟹猴。具体目标2：使用等基因基因敲除突变株来验证 四个选定的候选基因对GAS定殖和增殖的重要性，以及确定的持久性 全基因组筛选拟议的研究路线将利用我们的创新和成功 将TraDIS应用于GAS，包括食蟹猴感染。这将是第一次使用TRADIS 在雌性非人类灵长类动物的生殖道中， 使用全基因组转座子在粘膜表面上的定殖、增殖和持久性 诱变

PROJECT SUMMARY The human bacterial pathogen Streptococcus pyogenes (group A streptococcus, GAS) causes more than 700 million human infections annually worldwide. The proposed research seeks to discover, on a genome- wide scale, GAS genes required for, or contributing to fitness in the female genital tract, an infection site for which very little basic understanding is known. We will use a serotype M28 GAS strain because for reasons that aren’t understood, these organisms are non-randomly associated with female genital tract disease, including puerperal sepsis (childbed fever) and neonatal invasive infections. The pathogen genes and fundamental molecular pathogenesis processes contributing to two phases of genital tract interaction will be studied: (i) colonization and proliferation and (ii) persistence. This basic science knowledge deficit has severely limited the ability to create new clinical tools such as a successful GAS vaccine. Thus, the studies are designed to address this critical knowledge deficit. We will combine our very recent success in applying transposon-directed insertion site sequencing (TraDIS) technology to GAS with our 18 years of productive experience with cynomolgus macaque models of GAS infection, currently the gold standard experimental animal to study this human pathogen. We will use TraDIS to conduct an in vivo genome-wide screen that will systematically identify GAS genes required for colonization and proliferation, and persistence in the genital tract of female cynomolgus macaques. The goal of the proposed studies is to identify new molecular mechanisms used by GAS to enhance fitness in this anatomic niche. The results of our studies may ultimately help in formulating novel translational strategies to prevent or treat GAS infections. To achieve our goals, the following two specific aims are proposed: Specific Aim 1: Exploit TraDIS for genome-wide identification of GAS genes required for (i) colonization and proliferation, and (ii) persistence (i.e., fitness) in the genital tract of female cynomolgus macaques. Specific Aim 2: Use isogenic gene-knockout mutant strains to validate the importance of four selected candidate genes to GAS colonization and proliferation, and persistence identified by the genome-wide screen. The proposed line of research will exploit our innovative and successful application of TraDIS to GAS, including infections in cynomolgus macaques. It will be the first use of TraDIS in the genital tract of female non-human primates and the first study to investigate determinants of GAS colonization and proliferation, and persistence, on a mucosal surface using genome-wide transposon mutagenesis.