RpoS Regulation of Borrelia burgdorferi Genes in vivo

伯氏疏螺旋体是莱姆病(LD)的病原体，在自然界中存在于一种复杂的地方病中。 包括哺乳动物宿主和壁虱媒介的循环。为了维持这一循环，BB必须调整其 转录组、蛋白质组和代谢组到节肢动物和哺乳动物的宿主信号的穿梭 在这两者之间。RRP2/RpoN/rpos通路已被广泛认为是 破伤风基因调控。我们的实验室长期以来一直处于最前沿的努力，以确定队列的特征 扁虱和哺乳动物的rpos基因都受其控制。不幸的是，我们仍然不知道 RPOS-RNA聚合酶(RNAP)是自然界中的一种全酶。本提案通过以下方式解决这一短缺问题 结合最先进的转录和突变方法来确定个人在哪里/何时 基因有助于RPOS的双重宿主“任务”。在以前的出版物中，我们在描绘 活体内rpos开启和关闭状态的时间边界。然而，近年来，我们对此的思考 两分法已经变得更加微妙。我们现在相信rpos规则的轮廓发生了变化。 在Rpos-on期间以特定于宿主的方式深刻地陈述这些结果，与对 HK1/Rrp1途径，提出了我们的中心假说--rpos调节子的动态性质反映了 非RPOS调节通路与控制RPOS和 Rpos-rnap的输出。沿着这些思路，我们现在提出滴答阶段的信号分子c-di-GMP是 对这种监管方面的相互影响至关重要。此外，我们和其他人发现c-di-GMP结合蛋白Plza 促进rpos的表达，从而促进小鼠的Bb毒力，在地方性循环的阶段， HK1/Rrp1通路关闭。我们努力澄清PlzA作为c-di-GMP信号介体的这一双功能角色 在扁虱和小鼠体内不依赖c-di-GMP的rpos调节器中，将我们的领域带入了未知的领域。最后， 在过去的几年里，我们对在透析膜中培养的哺乳动物宿主适应螺旋体进行了分析 揭示了rpos不仅上调了蜱类传播和哺乳动物感染所需的基因，而且还 抑制定植和适应扁虱所需的基因。我们最近的研究表明，RPOS-RNAP 通过阻断其σ70启动子，直接抑制滴答期基因的转录。我们将此`RpoS测试为 抑制者模型及c-di-GMP拮抗rpos介导的哺乳动物抑制的新数据 寄主适应的BB。我们的长期目标是全面了解 RRP2/RpoN/rpos通路完成其基本使命--引导LD螺旋体从扁虱到小鼠再回到小鼠 再来一次。我们将通过定义扁虱和小鼠的rpos调节子的轮廓来实现这一点(目标1)；澄清 依赖c-di-GMP和Plza的信号与rpos通路的融合(目标2)；和解剖 哺乳动物宿主适应BB中rpos介导的滴答期基因抑制及c-di-GMP的拮抗作用 (目标3)。

Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), is maintained in nature within a complex enzootic cycle involving a mammalian reservoir host and a tick vector. To sustain this cycle, Bb must adjust its transcriptome, proteome, and metabolome to arthropod- and mammalian host-derived signals as it shuttles between the two. The Rrp2/RpoN/RpoS pathway has gained widespread recognition as a central player in borrelial gene regulation. Our laboratory has long been at the forefront of efforts to characterize the cohort of genes controlled by RpoS in both the tick and mammal. Unfortunately, we still do not know the entire output of RpoS-RNA polymerase (RNAP) holoenzyme in nature. The present proposal addresses this shortfall by combining state-of-the-art transcriptomic and mutagenesis methodologies to determine where/when individual genes contribute to RpoS's dual-host `mission'. In prior publications, we were instrumental in delineating the temporal boundaries of the RpoS-ON and –OFF states in vivo. In recent years, however, our thinking about this dichotomy has become much more nuanced. We now believe that the contours of the RpoS regulon change profoundly and in host-specific fashion during the RpoS-ON state These results, in concert with studies of the Hk1/Rrp1 pathway, give rise to our central hypothesis--the dynamic nature of the RpoS regulon reflects the confluence of non-RpoS regulatory pathways with mechanisms that govern the ON/OFF states of RpoS and the output of RpoS-RNAP. Along these lines, we now propose that the tick-phase signaling molecule c-di-GMP is crucial to this regulatory cross-talk. Moreover, we and others have found that the c-di-GMP-binding protein PlzA promotes expression of RpoS and, hence, Bb virulence in the mouse, the stage of the enzootic cycle in which the Hk1/Rrp1 pathway is OFF. Our efforts to clarify this bifunctional role of PlzA as a mediator of c-di-GMP signaling in ticks and a c-di-GMP-independent regulator of RpoS in mice takes our field into uncharted territory. Lastly, in years past, our analysis of mammalian host-adapted spirochetes cultivated in dialysis membrane chambers revealed that RpoS not only upregulates genes required for tick transmission and mammalian infection but also represses genes required for colonization and adaptation to the tick. Our recent work suggests that RpoS-RNAP directly represses transcription of tick-phase genes by occluding their σ70 promoters. We will test this `RpoS as repressor' model and explore new data that c-di-GMP antagonizes RpoS-mediated repression in mammalian host-adapted Bb. Our long-term objective is to achieve an integrative understanding of how the Rrp2/RpoN/RpoS pathway fulfills its essential mission--guiding LD spirochetes from tick to mouse and back again. We will accomplish this by defining the contours of the RpoS regulon in ticks and mice (Aim 1); clarifying the convergence of c-di-GMP- and PlzA-dependent signaling with the RpoS pathway (Aim 2); and dissecting RpoS-mediated repression of tick-phase genes and its antagonism by c-di-GMP in mammalian host-adapted Bb (Aim 3).