Effect of Mucins and Dolosigranlulum pigrum on Staphylococcus aureus nasal colonization

降低S的关键障碍。金黄色葡萄球菌感染正在确定非抗生素方法，以可靠地防止S。金黄色 鼻腔定植没有针对S的疫苗。aureus和S.金黄色葡萄球菌是主要的健康负担。第三 成年人有S。金黄色葡萄球菌鼻腔定植，这是一个危险因素，发展严重感染，在遥远的 身体部位感染，其中约80%的时间是由个体的鼻拉伤引起的。我的目标是解决 迫切需要新的，非抗生素的方法来预防S。通过鉴定宿主因子确定金黄色葡萄球菌鼻腔定植 （粘蛋白）和有益细菌（Dolosigranulum pigrum）具有预防S.金黄色鼻 殖民化高水平的D。pigrum与较低水平的S有关。金黄色葡萄球菌鼻腔定植 微生物群组成研究，和D. pigrum抑制S.金黄色葡萄球菌。采用创新模式 系统，人鼻上皮类器官（HNO），我已经成功地用每种细菌定殖HNO， 在生理鼻道温度下48小时。初步数据表明，D.皮格伦影响 MUC2的上皮表达，MUC2是一种与肠道中细菌耐受性相关的粘蛋白。HNO产生厚厚的 由功能性多纤毛细胞循环的粘液层。我的首要假设是D。黑变 S.金黄色葡萄球菌定植在粘液覆盖的鼻上皮的背景下。粘液是粘膜损伤的关键因素， 细菌定植;然而，其对S.金黄色葡萄球菌鼻内定殖知之甚少。我的目标是 以确定每种细菌如何影响粘液的产生，这又如何影响定植，以及是否 D. pigrum影响S. HNO的金黄色葡萄球菌定殖。在目标1中，我将定义D。pigrum和S.金黄色影响 通过定量定殖期间粘蛋白蛋白表达的HNO粘蛋白谱。我将决定殖民 当使用CRISPR的关键差异产生的粘蛋白存在与不存在时，每种细菌的成功- Cas9系统产生两个纯合突变HNO系，每个缺乏一种特异性粘蛋白。在目标2中，我将 确定如何D. pigrum影响S.金黄色葡萄球菌定殖和适合度。首先，我将描述 HNO上每种微生物单独和一起的免疫组织化学。然后定义S。金黄色葡萄球菌定植成功 通过1）定量CFU; 2）使用RNAseq比较其转录组;和3） 使用转座子突变体适合度分析（Tn-seq）与现有的S.金黄色葡萄球菌Tn-library鉴定基因 对HNO的适应性很重要。我将使用细菌遗传学来确定这些候选S。金黄色葡萄球菌基因 受D. pigrum和/或大大有助于健身对HNO。这个项目的意义在于， 它解决了关于鼻粘液产生如何影响细菌定植以及S. 金黄色葡萄球菌在复杂的粘液覆盖的上皮中响应宿主和细菌信号。这架F31会训练我 在一种新的鼻呼吸模型的背景下研究宿主-微生物和微生物-微生物的相互作用 上皮这将使我为实现我的长期目标做好准备，成为一名在 微生物-微生物相互作用、宿主-微生物相互作用和粘膜生物学的交叉。

A critical barrier to reducing S. aureus infections is identifying nonantibiotic methods to reliably prevent S. aureus nasal colonization. There is no vaccine against S. aureus, and S. aureus is a major healthcare burden. A third of adults have S. aureus nasal colonization, and this is a risk factor for developing serious infection at distant body sites, with an individual’s nasal strain responsible for infection ~80% of the time. My goal is to address the urgent need for new, nonantibiotic approaches to prevent S. aureus nasal colonization by identifying host factors (mucins) and beneficial bacteria (Dolosigranulum pigrum) with the potential to prevent S. aureus nasal colonization. High levels of D. pigrum are associated with lower levels of S. aureus nasal colonization in microbiota composition studies, and D. pigrum inhibits S. aureus on agar medium. Using an innovative model system, human nasal epithelial organoids (HNOs), I have successfully colonized HNOs with each bacterium for 48 hours at physiological nasal-passage temperature. My preliminary data shows that D. pigrum influences epithelial expression of MUC2, a mucin associated with bacterial tolerance in the gut. HNOs produce a thick mucus layer that is circulated by functional multiciliated cells. My overarching hypothesis is that D. pigrum alters S. aureus colonization in the context of a mucus-covered nasal epithelium. Mucus is a key factor in mucosal bacterial colonization; however, its impact on S. aureus nasal colonization is poorly understood. My objective is to determine how each bacterium affects mucus production, how this in turn influences colonization, and whether D. pigrum influences S. aureus colonization of HNOs. In Aim 1, I will define how D. pigrum and S. aureus affect HNO mucin profiles by quantifying mucin protein expression during colonization. I will determine colonization success by each bacterium when key differentially produced mucins are present vs. absent using the CRISPR- Cas9 system to generate two homozygous mutant HNO lines, each lacking one specific mucin. In Aim 2, I will determine how D. pigrum influences S. aureus colonization and fitness on HNOs. I will first characterize the biogeography of each organism alone and together on HNOs. I will then define S. aureus colonization success during mono- vs. cocolonization by 1) quantifying CFUs; 2) using RNAseq to compare its transcriptome; and 3) using transposon mutant fitness profiling (Tn-seq) with an existing S. aureus Tn-library to identify genes important for fitness on HNOs. I will use bacterial genetics to determine if 2 of these candidate S. aureus genes are influenced by D. pigrum and/or greatly contribute to fitness on HNOs. The significance of this project is that it addresses gaps in knowledge as to how nasal mucus production influences bacterial colonization and how S. aureus responds to host and bacterial signals in a complex, mucus-covered epithelium. This F31 will train me to investigate host-microbe and microbe-microbe interactions in the context of a new model of nasal respiratory epithelium. It will prepare me to achieve my long-term goal of becoming a Principal Investigator working at the intersection of microbe-microbe interactions, host-microbe interactions, and mucosal biology.