Host-Pathogen Interactions From Measurements of Urinary Cell Free DNA in Kidney Transplantation

肾移植是终末期肾病的首选治疗方案，但传染性和 免疫并发症限制了肾移植的寿命。尿路感染(UTI)发生在 肾移植后的发病率高得惊人，并且与尿毒症、同种异体移植物丢失和死亡率有关。 目前诊断尿路感染的金标准是细菌培养。然而，基于文化的测试受到以下因素的限制 它们只检测可培养细菌的能力，而不是告知混合感染和共生微生物区系，而是 不告知细菌生长动态，也不告知宿主对感染的反应。 这项提案的目标是发明并应用尿液无细胞DNA的基因组分析，从而能够 同时告知尿液微生物组的组成、病原体的生长动态 与尿路感染相关，移植物中的组织损伤，以及宿主对感染的免疫反应。独一无二的 这项工作的机会是我们超过250名肾移植受者的庞大队列，在这些队列中，我们有 移植前3个月的尿液标本和常规尿液培养。 使用一种新开发的单链DNA文库制备技术，我们将分析290个尿样 对于cfDNA：i)来自患有UTI的移植受者的序列样本(UTI之前、期间和之后)II)序列样本 来自反复尿路感染的受者；iii)无症状的尿路感染和尿液分析阴性的受者的样本； 移植后3个月内未发生尿路感染的受者的样本。 我们将对肾移植患者尿路感染前、中、后的动态尿菌群进行分析。 我们将调查cfDNA图谱是否与UTI相关，以及它们是否可以预测UTI， 尿路感染复发，耐多药尿路感染。基于细菌基因组复制率和 增长动力可以通过元基因组序列覆盖率的分析来估计，我们将估计增长 尿路感染和复发性尿路感染患者随时间和抗生素治疗反应的动态变化。 我们将通过分析cfDNA中包含的核小体足迹来监测宿主对UTI的反应 并通过供体特异的cfDNA。我们的初步研究表明：i)核小体阵列的结构 转录调控元件在尿液cfDNA中被保留和组成，以及ii)分析 核小体在基因启动子中的占有率可以用来量化基因表达。我们将调查 尿检阳性和无症状尿路感染患者的不同宿主反应 尿路感染患者在抗生素治疗后尿检呈阴性和宿主反应不同。我们会 以供体特异性cfDNA作为同种异体移植物损伤的标志物，在尿路感染范围内评估移植物的组织损伤。 这一建议的成功实施也将为尿路感染的发病机制提供新的理解 作为利用cfDNA分析作为敏感和非侵入性移植监测的新途径 工具。

Kidney transplantation is the preferred treatment option for end stage kidney disease, but infectious and immunological complications limit the lifespan of kidney transplants. Urinary tract infections (UTI) occur at an alarmingly high rate after kidney transplantation and are associated with urosepsis, allograft loss, and mortality. The current gold standard for diagnosis of UTI is bacterial culture. Culture-based tests are, however, limited by their ability to detect only culturable bacteria, do not inform about co-infections and commensal microbiota, do not inform about bacterial growth dynamics, and do not inform about the host's response to infection. The goal of this proposal is to invent and apply genomic assays of urinary cell-free DNA that can simultaneously inform about the composition of the urinary microbiome, growth dynamics of pathogens associated with UTI, tissue injury in the graft, and the host's immune response to infection. A unique opportunity for this work is our large cohort of over 250 kidney transplant recipients in which we have serial biobanked urine specimens as well as routine conventional urine cultures in the first 3 months of transplantation. Using a newly developed single-stranded DNA library preparation technique, we will profile 290 urinary samples for cfDNA: i) serial samples from transplant recipients with UTI (prior to, during, and after UTI) ii) serial samples from recipients with recurrent UTIs; iii) samples from recipients with asymptomatic UTI and negative urinanalysis; and iv) samples from recipients who do not develop UTI in the first 3 months of transplantation. We will profile the dynamic urinary microbiome prior to, during, and after UTI in kidney transplantation. We will investigate whether cfDNA profiles are associated with UTI as well as whether they can anticipate UTI, UTI recurrence, and multi-drug resistant UTI. Based on the concept that bacterial genome replication rates and growth dynamics can be estimated from analyses of metagenomic sequence coverage, we will estimate growth dynamics over time and in response to antibiotic therapy in patients with UTI and recurrent UTI. We will monitor the host response to UTI via analyses of nucleosome footprints comprised within cfDNA and via donor specific cfDNA. Our pilot studies indicate that i) the structures of nucleosomal arrays in transcriptional regulatory elements are preserved and comprised within urinary cfDNA, and ii) that an analysis of nucleosomal occupancy in gene promoters can be used to quantify gene expression. We will investigate the differential host's response in subjects who have symptomatic UTI with positive urinalysis and asymptomatic UTI with negative urinalysis and the differential host's response in UTI patients after antibiotic treatment. We will assess tissue injury to the allograft in the scope of UTI, using donor specific cfDNA as a marker of allograft injury. Successful implementation of this proposal will provide a novel understanding of the pathogenesis of UTI as well as open new avenues for the utilization of cfDNA profiling as a sensitive and noninvasive transplant monitoring tool.