In Host Evolution of Nontuberculous Mycobacteria in Cystic Fibrosis

项目总结 非结核分支杆菌(Ntm)已成为引起严重肺部疾病的一种日益常见的原因。 在囊性纤维化(CF)患者中。不幸的是，诊断、预后和治疗仍然非常困难。 很难。感染可能会导致肺功能更快地下降，甚至死亡。在慢性感染期间， NTM经常对最有效的抗生素方案产生抗药性，感染通常难以或 无法根除。此外，对致病的分子决定因素知之甚少。 炎症、免疫逃避和NTM驱动的疾病的持久性。在这里，我们提出一种方法，即 跟踪慢性感染CF患者中NTM的基因组随时间的演变。我们将严格执行 全基因组测序和一系列系统学分析以确定非结核分枝杆菌菌株的基因变化 这可能会增加病原体的适合性。因为肺泡灌洗液中含有复杂而动态的微生物 我们还将全面评估共生微生物区系，包括众所周知的CF病原体 以及呼吸道微生物区系的其他成员。虽然一些微生物区系可能与NTM竞争，但其他微生物区系可能 加强NTM感染，有些甚至可以作为有益于 病原体(例如，抗生素耐药性)。我们假设NTM经历了可预测的表型和 可能受到共同感染微生物区系影响的基因类型变化，以允许增强NTM 肺脏的适合性、毒性和持久性。目的1检查慢性萎缩性胃炎患者的纵向分离株 感染NTM，强调深度抽样方法和微生物种群水平估计 多样性、突变、重组和自然选择。NTM种群的进化变化将是 与详细的临床和表型信息相关联，以识别可能与抗生素有关的基因 抗药性、毒性和坚持性。Aim 2使用一种新的、完整的16S-rRNA基因测序方法来 检查NTM与其他肺微生物区系的横截面和纵向相互作用 感染CF病原体。我们将比较患有和不患有NTM的患者，并在 微生物种群。完成这些目标将确定可以作为靶点的基因和微生物相互作用 未来旨在揭示NTM的基本机制和策略的功能研究 寄主内进化过程中的适应。我们还将测试和验证生物信息管道和采样 策略，可用于未来的NTM系统基因组研究，我们将获得一个细粒度的理解 慢性感染期间非结核分枝杆菌的种群动态。表征宿主内NTM适应将使我们能够 更有效地识别可能难以根除或治疗的菌株，并设计新的、有针对性的 旨在颠覆这些适应的干预措施。

PROJECT SUMMARY Nontuberculous mycobacteria (NTM) have emerged as an increasingly common cause of severe lung disease in patients with cystic fibrosis (CF). Unfortunately, diagnosis, prognosis, and treatment remain exceedingly difficult. Infection can result in more rapid decline in lung function and even death. During chronic infection, NTM frequently become resistant to the most effective antibiotic regimens, and infection is typically difficult or impossible to eradicate. Moreover, little is known about the molecular determinants of pathogenesis, inflammation, immune evasion, and persistence in NTM-driven disease. Here we propose an approach that follows the genomic evolution of NTM over time in chronically infected CF patients. We will rigorously apply whole genome sequencing and a battery of phylogenomic analyses to identify genetic changes in NTM strains that are likely to increase pathogen fitness. Because the CF lung harbors a complex and dynamic microbial ecosystem we will also comprehensively assess co-inhabiting microbiota, including well-known CF pathogens and other members of the respiratory microbiota. While some microbiota may compete with NTM, others may act to reinforce NTM infections, and some could even serve as reservoirs for genes that are beneficial to the pathogen (e.g., antibiotic resistance). We hypothesize that NTM undergoes predictable phenotypic and genotypic changes, which are likely influenced by co-infecting microbiota, to allow for enhanced NTM fitness, virulence, and persistence in the lung. AIM 1 examines longitudinal isolates from CF patients infected with NTM, emphasizing a deep sampling approach and population-level estimates of microbial diversity, mutation, recombination, and natural selection. Evolutionary changes in NTM populations will be associated with detailed clinical and phenotypic information, to identify genes that may be involved in antibiotic resistance, virulence, and persistence. AIM 2 uses a novel, whole 16S-rRNA gene sequencing approach to examine the cross-sectional and longitudinal interaction of NTM with other lung microbiota including co- infecting CF pathogens. We will compare patients with and without NTM and associate differences in the microbial population. Completing these aims will identify genes and microbial interactions that can be targeted for future functional studies aimed at uncovering the basic mechanisms and strategies that allow NTM adaptation during in-host evolution. We will also test and validate bioinformatic pipelines and sampling strategies that can be used in future NTM phylogenomic studies, and we will gain a fine-grained understanding of the NTM population dynamics during chronic infection. Characterizing in-host NTM adaptation will allow us to more effectively identify strains that may be difficult to eradicate or treat, and design new, targeted interventions to subvert these adaptations.!