Characterization of C02 Sensing and Regulatory Response in Borrelia burgdorferi

描述（由申请人提供）：莱姆病的病原体伯氏疏螺旋体在节肢动物载体和哺乳动物宿主之间移动时适应不同的环境，这是公认的。B。burgdorferi调节基因表达和蛋白质合成以响应环境线索温度、pH、O2、CO2和其它未知宿主因素。CO2是细胞呼吸的副产品，其水平在哺乳动物宿主内波动，可能向病原生物体发出信号以进行宿主适应性反应。病原体利用CO2和HCO 3检测来激活毒素表达或诱导cAMP信号传导途径。我们假设B. Burgdorferi采用类似的机制以充分适应哺乳动物宿主中的发病机制。 我们的初步数据表明，CO2是向B发出的信号。burgdorferi来调节转录调节因子的产生和对感染性重要的基因的表达。两个疏螺旋体调节系统，Rrp 2-RpoN-RpoS调节系统和BosR蛋白，改变响应CO2表明这种环境线索是至关重要的B。burgdorferi的感觉，适应，并在哺乳动物宿主中生存。BosR，疏螺旋体氧化应激调节因子，是转录后调节响应CO2和相互作用的rpoS启动子的潜力，导致CO2的rpoS和RpoS依赖性基因的转录调控。为此，我们提出以下具体目标：（1）确定B使用的机制。（2）鉴定感染性B中受CO2调控的基因和蛋白。burgdorferi。在拟议的研究中，推定的疏螺旋体腺苷酸环化酶cyaB已被删除，并将评估相对于亲株的CO2调节以及对cAMP积累的抑制。我们将确定cyaB活性如何影响B的感染性。burgdorferi通过生物发光体内成像。最近 我们已经使用体内成像来检测光发射（即，荧光素酶[luc]表达）感染B。实验性感染后的伯氏螺旋体。这种方法的优点是B。随着时间的推移，在活小鼠中可以多次观察到伯氏菌，以跟踪感染过程。这种令人兴奋的方法提供了一个强大的非侵入性，真实的时间模式，以评估感染性的时间和空间的方式。此外，转录组学和蛋白质组学技术将用于产生对CO2有反应的基因和蛋白质的全面子集，其长期目标是为鉴定新的毒力决定因素和潜在的治疗靶点提供平台。 一种由B感知的直接检测环境线索的机制。Burgdorferi尚未被定性。本文提出的工作将表征CO2传感如何有助于B参与的复杂调节。burgdorferi在其复杂的生命周期中的作用。

DESCRIPTION (provided by applicant): It is well established that Borrelia burgdorferi, the etiologic agent of Lyme disease, adapts to distinct environments as it moves between an arthropod vector and mammalian hosts. B. burgdorferi modulates gene expression and protein synthesis in response to environmental cues temperature, pH, O2, CO2 and other unknown host factors. CO2 is a byproduct of cellular respiration and levels fluctuate within the mammalian host potentially signaling to pathogenic organisms for host adaptive responses. Pathogens utilize CO2 and HCO3 detection for activation of toxin expression or induction of a cAMP signaling pathway. We hypothesize that B. burgdorferi employs similar mechanisms to adequately adapt for pathogenesis in the mammalian host. Our Preliminary Data suggests CO2 serves as a signal to B. burgdorferi to regulate the production of a transcriptional regulator and the expression of genes that are important for infectivity. Two borrelial regulatory systems, the Rrp2-RpoN-RpoS regulatory system and the BosR protein, are altered in response to CO2 suggesting that this environmental cue is critical for B. burgdorferi to sense, adapt, and survive in the mammalian host. BosR, borrelial oxidative stress regulator, is post-transcriptionally regulated in response to CO2 and interacts with the rpoS promoter potential resulting in CO2 transcriptional regulation of rpoS and RpoS-dependent genes. To this end we propose the following Specific Aims: (1) Ascertain the mechanism utilized by B. burgdorferi to sense and respond to environmental CO2; (2) Identify genes and proteins regulated by CO2 in infectious B. burgdorferi. In the proposed studies, the putative borrelial adenylate cyclase, cyaB, has been deleted and the CO2 regulation will be evaluated, as well as inhibition of cAMP accumulation, relative to the parent strain. We will determine how cyaB activity contributes to infectivity of B. burgdorferi via bioluminescence in vivo imaging. Recently we have used in vivo imaging to detect light emitting (i.e., luciferase [luc] expressing) infectiou B. burgdorferi following experimental infection. The advantage of this approach is that B. burgdorferi can be visualized numerous times in live mice over time to track the infectious process. This exciting approach provides a powerful non-invasive, real time modality to evaluate infectivity in a temporal and spatial manner. In addition, transcriptomic and proteomic technologies will be utilized to generate a comprehensive subset of genes and proteins that are responsive to CO2 with the long term goal that this will provide a platform to identify novel virulence determinants and potential therapeutic targets. A mechanism of direct detection of an environmental cue that is sensed by B. burgdorferi has not yet been characterized. The proposed work herein will characterize how CO2 sensing contributes to the complex regulation engaged by B. burgdorferi as it traverses through its complex life cycle.