Characterization of C02 Sensing and Regulatory Response in Borrelia burgdorferi

描述(申请人提供)：众所周知，伯氏疏螺旋体是莱姆病的病原体，当它在节肢动物媒介和哺乳动物宿主之间移动时，能适应不同的环境。伯氏杆菌对环境信号温度、pH、O2、CO2和其他未知寄主因子的反应调节基因表达和蛋白质合成。二氧化碳是细胞呼吸的副产品，在哺乳动物宿主内水平波动，潜在地向病原体发出信号，以实现宿主的适应性反应。病原体利用CO2和HCO3检测来激活毒素表达或诱导cAMP信号通路。我们假设伯氏杆菌使用类似的机制来充分适应哺乳动物宿主的发病。 我们的初步数据表明，二氧化碳是伯氏杆菌的一个信号，可以调节转录调节因子的产生和对传染性至关重要的基因的表达。两个稀有的调控系统，RRP2-RpoN-rpos调控系统和BosR蛋白，对二氧化碳的响应发生了改变，这表明这一环境信号对伯氏杆菌在哺乳动物宿主中的感知、适应和生存至关重要。BOSR是一种氧化应激调节剂，受CO2的转录后调控，并与rpos启动子相互作用，导致CO2对rpos和rpos依赖基因的转录调控。为此，我们提出了以下具体目标：(1)确定伯氏杆菌感知和响应环境二氧化碳的机制；(2)鉴定感染性伯氏杆菌中受二氧化碳调控的基因和蛋白质。在拟议的研究中，假定的硼酸腺苷环化酶cyaB已被删除，并将评估相对于亲本菌株的二氧化碳调节以及对cAMP积累的抑制。我们将通过体内生物发光成像来确定cyaB活性如何影响伯氏杆菌的感染性。最近 我们已经使用体内成像来检测实验感染后的发光(即荧光素酶[Luc]表达)伯氏杆菌感染。这种方法的优点是，随着时间的推移，伯氏杆菌可以在活着的小鼠身上多次可视化，以跟踪感染过程。这一令人兴奋的方法提供了一种强大的、非侵入性的、实时的方式来以时间和空间的方式评估传染性。此外，还将利用转录学和蛋白质组学技术来产生对二氧化碳有反应的基因和蛋白质的综合子集，长期目标是提供一个平台来确定新的毒力决定因素和潜在的治疗靶点。 伯氏杆菌感受到的环境线索的直接检测机制尚未被表征。这里拟议的工作将描述二氧化碳传感如何有助于伯氏杆菌在其复杂的生命周期中进行复杂的调节。

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.