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

项目摘要/摘要。 金黄色葡萄球菌和铜绿假单胞菌是最常见的两种细菌 囊性纤维化(CF)合并慢性肺部感染。虽然历史上金黄色葡萄球菌通常被隔离在 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 在所有慢性萎缩性胃炎患者的痰标本中检测到。因此，存在很大的时间间隔，当S和S。 在CF痰标本中同时发现金黄色葡萄球菌和铜绿假单胞菌。多项研究，包括来自 我们的团队已经证明，合并感染与肺功能减弱和肺功能下降有关， 然而，这种不良结局的分子机制尚不清楚。虽然大多数报告都指出，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。 与金黄色葡萄球菌共存。关于金黄色葡萄球菌和对CF肺的适应，但出现除外 小菌落变异和粘液表型，这是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CFs呼吸丧失的病例 功能--还不能很好地理解。我们假设有更多的金黄色葡萄球菌基因 在CF肺环境中，在铜绿假单胞菌存在的情况下促进生存的决定因素。至 为了验证这一假设，我们开发了一种新的共培养方法，并测试了65株沙门氏菌的CF分离物。 金黄色葡萄球菌(来自50名患有CF的个体)，取自Emory CF Biospecimen Repository(CF-BR)和 波士顿儿童医院与非粘液型铜绿假单胞菌实验室菌株PAO1和PAO1的相互作用 它的粘液衍生物，并根据这些相互作用将这些分离物分为3组。这些 分离株已经进行了全基因组测序(WGS)，我们有与 采集标本时的CF患者。在这里，我们将采取三种互补的方法来确定 促进金黄色葡萄球菌与铜绿假单胞菌相互作用的遗传因素。我们将获得并排序~200 额外的纵向金黄色葡萄球菌分离株存放在Emory CF-BR中。特别是，我们将选择 在几年的样本采集和采集过程中，不同的铜绿假单胞菌混合感染状况 确定遗传相似菌株之间的SNP差异。我们将试验性地将金黄色葡萄球菌进化成 在铜绿假单胞菌存在的情况下存活。WGS与先祖相比将识别出负责 对铜绿假单胞菌的抗性。我们将对部分临床分离的金黄色葡萄球菌进行TN-seq分析。 共培养中铜绿假单胞菌的存在。在与铜绿假单胞菌相互作用后丢失的突变株将定义 在这些条件下对生存至关重要的基因。在这项研究结束时，我们将确定S. 使其与铜绿假单胞菌共存的金黄色葡萄球菌基因；这些因素的未来特征应该是 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。

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.