The RpoN-RpoS regulatory pathway in Borrelia burgdorferi

描述（由申请人提供）：莱姆病螺旋体伯氏疏螺旋体（Bb）在其不同蜱和哺乳动物宿主之间自然循环时经历显著的适应性变化。在主要研究者在过去的资助期间进行工作之前，对调节Bb的适应性变化的遗传调控网络知之甚少，这些变化有助于Bb的感染性和毒力。为此，我们在Bb中发现了一种新的调节途径，其中一种替代性σ因子（CN，RpoN）调节另一种替代性σ因子（Cs，RpoS）的表达，所述另一种替代性σ因子反过来支配与Bb的毒力相关的关键膜脂蛋白的表达。该途径由增强子结合蛋白，反应调节蛋白-2（Rrp 2）控制，其首先必须被激活以用于随后的RpoN激活和随后的rpoS转录。虽然在Bb中发现这种调控网络代表了莱姆病领域的一个重要进展，但它也引发了许多新的问题，涉及其直接和间接的调控作用，该途径如何与其他调控机制交叉，以及如何以及在何种程度上影响Bb的各种基因。在下一个供资期间，选定的调查领域将继续解决其中一些突出的信息差距问题。因此，本研究的具体目的是：（1）识别受RpoN-RpoS通路直接影响的基因。我们将联合收割机结合我们在Bb微阵列方面的专业知识和我们新开发的lac阻遏物/操纵子系统，人工控制Bb中rpoS的表达。目的4：（2）研究RpoN-RpoS通路调控核心蛋白聚糖结合蛋白（DbpB/A）操纵子非典型表达的机制。dbpBA操纵子在RpoN-RpoS途径的控制下，但其具有不同于由RpoS控制的其他基因的表达模式（例如，ospC）。因此，dbpBA可能是受该途径影响的某些基因如何受到另一层调控的模型。重点将放在分析dbpBA启动子和上游顺式元件的关键特征;（3）检查在Bb的整个地方性生活周期（蜱和哺乳动物阶段）的rpoS的表达;和（4）检查在选定的RpoS依赖基因缺陷的Bb突变体的表型。这些努力将为进一步表征Bb中的新型RpoN-RpoS调控网络提供一个不断扩展的基础，并为潜在地识别有助于Bb复杂寄生策略的许多方面的调控和毒力相关基因提供基础。了解这些基因的作用可能会导致莱姆病的新干预策略（疫苗，疗法）。

DESCRIPTION (provided by applicant): Borrelia burgdorferi (Bb), the Lyme disease spirochete, undergoes dramatic adaptive changes as it cycles in nature between its diverse tick and mammalian hosts. Prior to work conducted by the principle investigator over the past funding interval, little was known regarding the genetic regulatory networks that modulate Bb's adaptive changes contributing to Bb's infectivity and virulence. To this end, we discovered a novel regulatory pathway in Bb, wherein one alternative sigma factor (CN, RpoN) regulates the expression of another alternative sigma factor (Cs, RpoS) that, in turn, governs the expression of key membrane lipoproteins associated with Bb's virulence. The pathway is controlled by an enhancer-binding protein, response regulator protein-2 (Rrp2), which first must be activated for the subsequent activation of RpoN and the consequent transcription of rpoS. Although the discovery of this regulatory network in Bb has represented an important advance in the Lyme disease field, it also has prompted many new questions regarding its direct and indirect regulatory effects, how the pathway intersects with other regulatory mechanisms, and how and to what extents various genes of Bb are influenced. Selected areas of investigation over the next funding interval will continue to address some of these salient information gaps. Accordingly, the Specific Aims of this proposal are: (1) To discern those genes directly influenced by the RpoN-RpoS pathway. We shall combine our expertise in Bb microarrays with our newly developed lac repressor/operator system to artificially control the expression of rpoS in Bb. Identified genes will become the subject of further studies (Aim 4); (2) To investigate the mechanism by which the RpoN-RpoS pathway regulates atypical expression of the decorin-binding protein (DbpB/A) operon. The dbpBA operon is under the control of the RpoN-RpoS pathway, but it has an expression pattern different from other genes controlled by RpoS (e.g., ospC). As such, dbpBA may be a model of how certain genes influenced by the pathway also are subjected to another layer of regulation. Emphasis will be placed on analyzing key features of the dbpBA promoter and upstream cis-elements; (3) To examine the expression of rpoS over the complete enzootic life cycle (tick and mammalian phases) of Bb; and (4) To examine the phenotypes of Bb mutants deficient in selected RpoS-dependent genes. These efforts will provide an expanding foundation for further characterizing the novel RpoN-RpoS regulatory network in Bb, and for potentially identifying regulatory and virulence-associated genes that contribute to many aspects of Bb's complex parasitic strategy. Understanding the roles of these genes could lead to new intervention strategies (vaccines, therapeutics) for Lyme disease.