Mechanisms of Staphylococcus aureus and Pseudomonas aeruginosa Co-existence in CF

CF中金黄色葡萄球菌和铜绿假单胞菌共存的机制

基本信息

批准号：
10078252
负责人：
Joanna B Goldberg
金额：
$ 22.95万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10078252
关键词：
Bacteria Biological Assay Biological Specimen Banks Boston Chronic Clinical Coculture Techniques Cystic Fibrosis Cystic Fibrosis sputum Data Reporting Environment Foundations Future Genes Genetic Genetic Determinism Genome Goals Intrinsic factor Laboratories Lung Lung infections Metadata Molecular Molecular Genetics Outcome Patient Isolators Patient-Focused Outcomes Pediatric Hospitals Phenotype Pseudomonas aeruginosa Pulmonary Fibrosis Reporting Resistance Respiratory physiology Sampling Sequence Analysis Severity of illness Sputum Staphylococcus aureus Testing Therapeutic Time Variant base co-infection cystic fibrosis infection cystic fibrosis patients genome sequencing interest mucoid mutant pathogenic bacteria patient registry progenitor public health relevance pulmonary function time interval transposon sequencing whole genome

项目摘要

PROJECT SUMMARY/ ABSTRACT. Staphylococcus aureus and Pseudomonas aeruginosa are the two most common bacterial species associated with chronic lung infections in cystic fibrosis (CF). While historically S. aureus was commonly isolated in younger CF patients and then replaced by P. aeruginosa, it is now the bacterial pathogen most frequently detected in sputum samples from all CF patients. Consequently, there is a large time interval when both S. aureus and P. aeruginosa are found together in CF sputum samples. A number of studies including those from our group have shown that co-infection is associated with diminished lung function and pulmonary decline, however the molecular mechanism for this poor outcome is not known. While most reports have noted that P. aeruginosa readily kills S. aureus, we have found that the mucoid phenotype of P. aeruginosa aids in its co- existence with S. aureus. With respect to S. aureus and adaptation to the CF lung, other than the emergence of small colony variants and the mucoid phenotype, the factors intrinsic to S. aureus that promote chronic CF colonization—as well as those that engender co-existence with P. aeruginosa and elicit loss of CF respiratory function—are not well understood. We hypothesize that there are additional S. aureus genetic determinants that promote survival in the presence of P. aeruginosa in the CF lung environment. To begin to test this hypothesis, we have developed a new co-culture assay and tested 65 CF isolates of S. aureus (from 50 individuals with CF) obtained from the Emory CF Biospecimen Repository (CF-BR) and Boston Children's Hospital for their interactions with the non-mucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative and have categorized these isolates into 3 groups based on these interactions. These isolates have been subjected to whole genome sequencing (WGS) and we have the metadata associated with the CF patients at the time the samples were taken. Here we will take 3 complementary approaches to identify genetic factors of S. aureus that promote its interactions with P. aeruginosa. We will obtain and sequence ~200 additional longitudinal CF patient isolates of S. aureus banked in the Emory CF-BR. In particular, we will select isolates that differ in their P. aeruginosa co-infection status over several years of sample acquisition and identify SNP differences between genetically similar isolates. We will experimentally evolve S. aureus to survive in the presence of P. aeruginosa. WGS compared to the progenitor will identify genes responsible for resistance to P. aeruginosa. We will perform Tn-seq analysis of S. aureus on select clinical isolates in the presence of P. aeruginosa in co-culture. Mutants that are lost after interaction with P. aeruginosa will define genes that are critical for survival under these conditions. By the end of this study, we will have identified S. aureus genes that allow it to co-exist with P. aeruginosa; future characterization of these factors should increase our understanding of why co-infection correlates with worse patient outcome in CF.

项目摘要/摘要。金黄色葡萄球菌和铜绿假单胞菌是两种最常见的相关细菌种类囊性纤维化 (CF) 中的慢性肺部感染，而历史上的金黄色葡萄球菌通常在以下情况中分离出来。年轻的CF患者，然后被铜绿假单胞菌取代，它现在是最常见的细菌病原体在所有接受测试的 CF 患者的痰样本中均检测到，两者之间存在很大的时间间隔。许多研究都在 CF 痰样本中同时发现了金黄色葡萄球菌和铜绿假单胞菌。我们的研究小组已经证明，合并感染与肺功能下降和肺功能衰退有关，然而，这种不良结果的分子机制尚不清楚，尽管大多数报告都指出 P. 铜绿假单胞菌很容易杀死金黄色葡萄球菌，我们发现铜绿假单胞菌的粘液表型有助于其协同作用关于金黄色葡萄球菌的存在以及对 CF 肺的适应，除了出现之外。小菌落变异和粘液表型，金黄色葡萄球菌促进慢性 CF 的内在因素定植——以及那些与铜绿假单胞菌共存并导致 CF 呼吸丧失的定植我们还没有充分了解金黄色葡萄球菌的功能。在 CF 肺环境中铜绿假单胞菌存在下促进存活的决定因素。为了开始检验这一假设，我们开发了一种新的共培养测定法并测试了 65 个 CF 沙门氏菌分离株。金黄色葡萄球菌（来自 50 名 CF 患者）从埃默里 CF 生物样本库 (CF-BR) 获得，以及波士顿儿童医院与非粘液铜绿假单胞菌实验室菌株 PAO1 和其粘液衍生物，并根据这些相互作用将这些分离物分为 3 组。分离株已接受全基因组测序 (WGS)，我们拥有与以下相关的元数据采集样本时的 CF 患者在这里，我们将采取 3 种补充方法来识别。我们将获得并测序约 200 个金黄色葡萄球菌促进其与铜绿假单胞菌相互作用的遗传因素。我们将特别选择储存在 Emory CF-BR 中的额外纵向 CF 患者金黄色葡萄球菌分离株。经过几年的样本采集，其铜绿假单胞菌共感染状态有所不同的分离株识别遗传相似分离株之间的 SNP 差异我们将通过实验将金黄色葡萄球菌进化为在铜绿假单胞菌存在下存活，与祖细胞相比，WGS 将识别负责的基因。我们将对铜绿假单胞菌的选定临床分离株进行 Tn-seq 分析。与铜绿假单胞菌相互作用后丢失的突变体的存在将定义为共培养物中铜绿假单胞菌的存在。在这些条件下对生存至关重要的基因。允许其与铜绿假单胞菌共存的金黄色葡萄球菌基因应该是这些因素的未来特征；增加我们对为什么合并感染与 CF 患者预后较差相关的理解。