Persister cells of Borrelia burgdorferi

摘要 伯氏疏螺旋体是莱姆病的病原体， 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 短期抗生素治疗然而，如果允许进展到晚期症状， 对于关节炎或脑病，建议使用最多28天的更长疗程的抗生素。 即使使用更长的抗生素疗程，也有相当一部分莱姆关节炎患者会 没有改善，需要额外的治疗。鉴于抗生素耐药性 未观察到B。burgdorferi，晚期疾病复发的原因， 抗生素不清楚。在其他慢性感染中，耐药持续者的存在 与疾病的复发有关。初步研究发现，B. burgdorferi 形成耐药持续细胞，类似于由其他病原体形成的那些。在一个试点Tn-seq 在实验中，我们鉴定了参与B中持续存在形成的候选基因。burgdorferi 用头孢曲松治疗 这个项目的目标是确定在疏螺旋体持续形成的机制 burgdorferi，这将有助于了解它们在药物耐受性中的作用。学习E。大肠杆菌作为模型 生物，我们确定毒素/抗毒素（TA）模块作为形成的主要贡献者， 休眠的持留细胞然而，B。burgdorferi缺乏TA模块，这表明它进化了 药物耐受的独立机制。我们开发了几种互补的方法， 鉴定持久性基因，这些将应用于B的研究。burgdorferi。一是 使用Tn-seq扩展我们对影响持续存在形成的突变的初步鉴定， 其他临床相关抗生素。将对持续存在者进行转录组分析 通过用作用于细胞壁的抗生素裂解培养物来分离;以及通过在以下步骤中分选持留菌来分离。 一种代谢标志物通过离体筛选获得高持活力突变体（hip）， 通过全基因组测序鉴定突变基因。我们还将筛选髋关节突变， 野生蜱和病人，以了解这些是否可以解释疾病的变化 症状和对抗生素的反应。将优先考虑已确定的候选人 通过不同的方法。目的基因的缺失与过表达对细胞增殖的相反影响 药物耐受性将验证持续基因。对基因的详细检查将导致 全面了解耐药形成和耐药机制， 这种病原体，为更好的治疗方法的发展提供信息。

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.