Effect of Mucins and Dolosigranlulum pigrum on Staphylococcus aureus nasal colonization

减少金黄色葡萄球菌感染的一个关键障碍是确定可靠地预防金黄色葡萄球菌的非抗生素方法 鼻腔移居。目前还没有针对金黄色葡萄球菌的疫苗，金黄色葡萄球菌是主要的医疗负担。三分之一 的成年人有金黄色葡萄球菌鼻腔定植，这是远距离发展为严重感染的危险因素。 身体部位，约80%的时间是由个人的鼻部拉伤引起的。我的目标是解决 迫切需要新的非抗生素方法通过识别宿主因素来预防金黄色葡萄球菌鼻腔定植 (粘蛋白)和有可能预防金黄色葡萄球菌鼻腔的有益细菌(Dolosigranulum Pgrum) 殖民主义。高水平的粪肠球菌与较低水平的金黄色葡萄球菌鼻部定植有关 微生物区系组成研究，在琼脂上对金黄色葡萄球菌有抑制作用。使用创新模式 系统，人鼻上皮有机物(HNO)，我已成功地定植HNO与每种细菌 生理鼻腔温度48小时。我的初步数据显示，斑潜蝇会影响 肠道细菌耐受相关粘蛋白MUC2的上皮表达。HNO产生浓厚的 由功能性多纤毛细胞循环的粘液层。我的最重要的假设是，小球藻改变了 金黄色葡萄球菌在粘液覆盖的鼻黏膜上皮中的定植。粘液是粘膜中的一个关键因素 细菌定植；然而，它对金黄色葡萄球菌鼻部定植的影响却知之甚少。我的目标是 为了确定每种细菌如何影响粘液产生，这又如何影响定植，以及是否 金黄色葡萄球菌对HNO定植的影响。在目标1中，我将定义小金黄色葡萄球菌和金黄色葡萄球菌如何影响 HNO粘蛋白在定植过程中的定量表达。我将决定殖民 当关键的差异产生的粘蛋白存在或不存在时，每个细菌使用CRISPR- Cas9系统产生两个纯合子突变体HNO系，每个系都缺少一个特定的粘蛋白。在《目标2》中，我会 确定金黄色葡萄球菌如何影响金黄色葡萄球菌在HNO上的定植和适合性。我将首先描述一下 每种生物单独和共同在HNO上的生物地理学。然后我将定义金黄色葡萄球菌的定植成功 通过1)定量CFU；2)使用RNAseq比较其转录组；以及3) 利用转座子突变适合度分析(TN-SEQ)和现有的金黄色葡萄球菌TN文库鉴定基因 对HNO的健康很重要。我将使用细菌遗传学来确定这些候选金黄色葡萄球菌中的两个基因 和/或对HNO的适合性有很大贡献。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细胞中对宿主和细菌的信号做出反应。这辆F31将训练我 在一种新的鼻呼吸道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 上皮组织。这将为我实现成为一名首席调查员的长期目标做好准备 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.