Persister cells of Borrelia burgdorferi

摘要 伯氏疏螺旋体是莱姆病的病原体，据估计 在美国，每年有30万人。如果及早治疗，这种疾病通常很容易治愈。 使用短疗程的抗生素。然而，如果允许进展到晚期症状，如 对于关节炎或脑病，建议使用长达28天的抗生素疗程。 即使使用较长疗程的抗生素，相当大比例的莱姆关节炎患者也会 病情没有好转，需要额外的疗程。鉴于抗生素耐药性已经 未被观察到的伯氏杆菌病晚期顽固性的原因 抗生素目前还不清楚。在其他慢性感染中，耐药持久者的存在 与该病的顽固不化有关。在初步研究中，我们发现伯氏杆菌 形成耐药的持久细胞，类似于其他病原体形成的细胞。在试点TN-SEQ中 实验中，我们发现了与伯氏杆菌持续体形成有关的候选基因。 用头孢曲松治疗。 本项目的目标是确定疏螺旋体的持续体形成机制。 这将有助于理解它们在药物耐受性中的作用。以大肠杆菌为模型进行研究 生物体，我们发现毒素/抗毒素(TA)模块是形成 休眠的持久细胞。然而，B.burgdorferi缺乏TA模块，这表明它是进化的 药物耐受的独立机制。我们开发了几种免费的方法来 确定持久基因，这些基因将被应用于伯氏杆菌的研究。首先，我们将 使用TN-seq和扩展我们对影响永久形成的突变的初步鉴定 额外的临床相关抗生素。将对持久者进行转录组分析 通过用细胞壁起作用的抗生素裂解培养物并通过以下方式对宿根进行分选而分离 一种代谢标记物。将通过体外筛选获得高耐受性突变体(HIP)，并且 通过全基因组测序鉴定突变基因。我们还将筛选来自 了解这些因素是否可以解释疾病的变化 表现和对抗生素的反应。被确定的候选人将被优先考虑 通过不同的方法。目的基因缺失与过表达的相反影响 药物耐受性将验证一种持久基因。对基因的详细检查将导致 对耐药机制及耐药机制的全面认识 这种病原体，通知开发更好的治疗方法。

Abstract Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the US. When treated early, the disease is usually easily treated with short courses of antibiotics. However, if allowed to progress to late stage symptoms such as arthritis or encephalopathy, longer courses of up to 28 days of antibiotics are recommended. Even with longer courses of antibiotics, a significant proportion of patients with Lyme arthritis will not improve and will require additional courses of treatment. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In a preliminary study, we find that B. burgdorferi forms drug-tolerant persister cells similar to those formed by other pathogens. In a pilot Tn-seq experiment, we identified candidate genes involved in persister formation in B. burgdorferi treated with ceftriaxone. The goal of this project is to identify the mechanisms of persister formation in Borrelia burgdorferi, which will help understand their role in drug tolerance. Studying E. coli as a model organism, we identified toxin/antitoxin (TA) modules as a major contributor to formation of dormant persister cells. However, B. burgdorferi lacks TA modules, suggesting that it evolved independent mechanisms of drug tolerance. We developed several complimentary methods to identify persister genes, and these will be applied to the study of B. burgdorferi. First, we will expand our initial identification of mutations that affect persister formation using Tn-seq with additional clinically relevant antibiotics. Transcriptome analysis will be performed on persisters isolated by lysing the culture with a cell wall acting antibiotic; and by sorting persisters following a metabolic marker. High-persister mutants (hip) will be obtained by selection in vitro, and the mutant genes identified by whole genome sequencing. We will also screen for hip mutants from wild ticks and patients to understand if these could account for variations in disease manifestation and response to antibiotics. Priority will be given to candidates that are identified by different approaches. Opposite effects of deletion vs. overexpression of a gene of interest on drug tolerance will validate a persister gene. A detailed examination of the genes will lead to a comprehensive understanding of the mechanism of persister formation and drug tolerance in this pathogen, informing development of better treatments.