Effect of Mucins and Dolosigranlulum pigrum on Staphylococcus aureus nasal colonization

粘蛋白和猪白粉对金黄色葡萄球菌鼻定植的影响

基本信息

批准号：
10678143
负责人：
Andrea Ivey Boyd
金额：
$ 4.77万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10678143
关键词：
Address Adult Affect Agar Antibiotics Antimicrobial Resistance Bacteria Behavior Biological Models Biology CRISPR/Cas technology Carbon Cell Culture Techniques Cells Collaborations Colony-forming units Complex Corynebacterium Data Data Analyses Distant Environment Enzyme-Linked Immunosorbent Assay Epithelium Experimental Designs Fluorescent in Situ Hybridization Genes Genetic Genetic Transcription Goals Growth Healthcare Hospitalization Host Defense Hour Human Individual Infection Infection prevention Integration Host Factors Knowledge Learning Libraries Literature MUC5AC gene MUC5B gene Methods Microbe Microscopy Modeling Mucin-2 Staining Method Mucins Mucous Membrane Mucous body substance Nasal Epithelium Nose Organism Organoids Pathogenesis Pathogenicity Phenotype Physiological Piedra Prevention Principal Investigator Process Production Proteins Research Risk Factors Signal Transduction Signaling Molecule Site Source Staphylococcus aureus Staphylococcus aureus infection System Techniques Temperature Testing Thick Time Training Transcript Vaccines Virulence Factors airway epithelium bacterial genetics care burden cell immortalization expectation fitness gut bacteria host-microbe interactions innovation insight member microbiota mutant nasal microbiota pathobiont prevent protein expression skills success transcriptome transcriptome sequencing

项目摘要

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.

减少金黄色葡萄球菌感染的一个关键障碍是确定可靠预防金黄色葡萄球菌的非抗生素方法没有针对金黄色葡萄球菌的疫苗，金黄色葡萄球菌是一个主要的医疗负担。的成年人鼻腔有金黄色葡萄球菌定植，这是远处发生严重感染的危险因素身体部位，大约 80% 的情况是由个人的鼻部劳损引起的，我的目标是解决这个问题。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定植（粘蛋白）和有益细菌（Dolosigrinum Pigrum）具有预防金黄色葡萄球菌鼻腔的潜力高水平的猪 D. 猪与较低水平的金黄色葡萄球菌鼻腔定植相关。微生物群组成研究，猪 D. Pigrum 使用创新模型抑制琼脂培养基上的金黄色葡萄球菌。系统，人鼻上皮类器官（HNO），我已经成功地将每种细菌定植于 HNO 上在生理鼻道温度下 48 小时我的初步数据表明 D. Pigrum 会产生影响。 MUC2（一种与肠道细菌耐受性相关的粘蛋白）的上皮表达会产生粘稠的粘液。我的总体假设是 D. Pigrum 改变了由功能性多纤毛细胞回收的粘液层。金黄色葡萄球菌在粘液覆盖的鼻上皮中定植是粘膜的关键因素。细菌定植；然而，它对金黄色葡萄球菌鼻定植的影响却知之甚少。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 D. Pigrum 影响 S. aureus 在 HNO 中的定殖在目标 1 中，我将定义 D. Pigrum 和 S. aureus 如何影响。我将通过量化定植过程中粘蛋白蛋白的表达来确定 HNO 粘蛋白谱。当关键的差异产生的粘蛋白存在与不存在时，每种细菌使用 CRISPR 所取得的成功 Cas9 系统生成两个纯合突变体 HNO 系，每个系都缺乏一种特定的粘蛋白。在目标 2 中，我将。确定 D. Pigrum 如何影响金黄色葡萄球菌在 HNO 上的定植和适应度。然后，我将通过对 HNO 上每种生物体单独和共同的生物地理学来定义金黄色葡萄球菌的定殖成功。在单定植与共定植期间，通过 1) 定量 CFU；2) 使用 RNAseq 比较其转录组；使用转座子突变体适应性分析 (Tn-seq) 和现有的金黄色葡萄球菌 Tn 文库来识别基因对于 HNO 的适应性很重要，我将使用细菌遗传学来确定这些候选金黄色葡萄球菌基因中是否有 2 个。受到 D. Pigrum 的影响和/或对 HNO 的适应性有很大贡献该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金黄色葡萄球菌如何影响细菌定植的知识空白。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信号做出反应。这个 F31 将训练我在新型鼻呼吸模型的背景下研究宿主-微生物和微生物-微生物之间的相互作用它将使我做好准备，实现成为一名首席研究员的长期目标。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点。