A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions.
A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions.
复制标题
DOI：
10.3389/fcimb.2017.00225
复制
发表时间：
2017
影响因子：
5.7
通讯作者：
Jewett MW
中科院分区：
文献类型：
--
作者：
Adams PP;Flores Avile C;Jewett MW
了解病媒传播病原体在病媒-病原体界面上的转录反应对于了解疾病传播至关重要。伯氏疏螺旋体是美国莱姆病的病原，主要通过被感染的硬蜱叮咬传播。滴答滴答。据了解，伯氏伯氏杆菌在蜱虫的获取、持续和传播过程中改变了基因表达模式。最近，我们和其他人发现了在伯氏杆菌基因组中发现的内部、重叠和反义的RNA的体外表达。然而，伯氏杆菌缺乏分子遗传学工具来在不同的环境中对这些转录本进行定量、链特异性和比较分析，例如节肢动物媒介。为了满足这一需求，我们开发了一种双重荧光素酶报告系统，以链特异性的方式量化伯氏杆菌启动子的活性。我们证明了当与伯氏杆菌密码子优化的石楠荧光素酶基因(FlucBb)在同一质粒上融合时，伯氏杆菌密码子优化的肾氏杆菌荧光素酶基因(RLucBb)的组成性表达允许目标启动子的活性标准化。通过对鞭毛蛋白启动子(Fla Bp)、外表面蛋白A(OspAp)和外表面蛋白C(OspCp)启动子的研究，我们证明了双重荧光素酶系统在体外生长过程中和在受感染的硬体中定量检测启动子活性的有效性。总而言之，本文概述的双荧光素酶方法是伯氏假单胞菌的第一个双报告系统，为链特异性分子遗传分析提供了一种新的和高度通用的方法。
Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rlucBb) allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (flucBb) on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp), outer surface protein A (ospAp) and outer surface protein C (ospCp), we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses.