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

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

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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关键障碍是识别非抗生素方法，以可靠地预防金黄色葡萄球菌鼻定植。没有针对金黄色葡萄球菌的疫苗，而金黄色葡萄球菌是主要的医疗保健负担。第三成年人有金黄色葡萄球菌鼻定植，这是在远处发展严重感染的危险因素身体部位，有个人的鼻腔菌株，导致感染约80％。我的目标是解决迫切需要通过识别宿主因素来防止金黄色葡萄球菌鼻定植的新的，非抗生素的方法（粘蛋白）和有益细菌（dolosigranulum pigrum），有可能预防金黄色葡萄球菌殖民化。高水平的小斑杆菌与较低水平的金黄色葡萄球菌鼻定植有关微生物群的组成研究，而D. pigrum在琼脂培养基上抑制金黄色葡萄球菌。使用创新模型系统，人类鼻上皮器官（HNOS），我已经成功地与每种细菌定殖的HNO 在物理鼻腔通风温度下48小时。我的初步数据表明D. pigrum的影响 MUC2的上皮表达，一种与肠道中细菌耐受性相关的粘蛋白。 HNO会产生厚粘液层由功能多细胞循环。我的总体假设是D. pigrum改变了在粘液覆盖的鼻上皮的背景下，金黄色葡萄球菌定植。粘液是粘膜的关键因素细菌定殖；然而，它对金黄色葡萄球菌鼻定植的影响知之甚少。我的目标是为了确定每种细菌如何影响粘液产生，这又如何影响定殖以及是否存在 D. Pigrum影响HNO的金黄色葡萄球菌定植。在AIM 1中，我将定义D. pigrum和S.金黄色葡萄球菌如何影响 HNO粘蛋白特征通过定量定植过程中的肌蛋白蛋白表达。我将确定殖民化当关键产生不同产生的粘蛋白时，每个批处理的成功与使用CRISPR-存在不同 CAS9系统生成两个纯合突变型线，每种缺乏一个特定的突变。在AIM 2中，我会确定D. pigrum如何影响金黄色葡萄球菌定植和适应性对HNOS。我将首先描述单独和HNO的每个生物的生物地理学。然后，我将定义金黄色葡萄球菌定殖成功通过1）量化cfus;在单声道中与co殖民化期间； 2）使用RNASEQ比较其转录组； 3）使用现有的金黄色葡萄球菌TN-图鉴定基因对于HNO的健身至关重要。我将使用细菌基因来确定这些候选金黄色葡萄球菌是否中的2个受d。pigrum的影响和/或极大地有助于HNO的适应性。该项目的重要性是它解决了知识的差距，即鼻粘液产生如何影响细菌定植以及S. 金黄色葡萄球菌对复杂的，粘液覆盖的上皮中的宿主和细菌信号反应。这个F31会训练我在新的鼻呼吸模型的背景下，研究宿主微杆和微生物 - 微生物相互作用上皮。这将使我能够实现我成为在工作的主要研究者的长期目标微生物 - 微生物相互作用，宿主微叶相互作用和粘膜生物学的交点。