The Roles of RpoS and the Borrelia Oxidative Stress Regulator, BosR, in the Transmission of Relapsing Fever Spirochetes

该项目正在研究赫氏疏螺旋体中关键毒力因子的调节，这些因子是成功地从其媒介鸟鼻向哺乳动物宿主传播所必需的。虽然赫氏杆菌具有与伯氏杆菌相似的调控蛋白基因(即rpos、rpoD和疏螺旋体氧化应激调节因子BosR)，但该细菌对环境信号的反应，如温度、活性氧(ROS)、活性氮(RNS)、溶解氧和pH，都有很大的不同。这些差异反映了这些致病螺旋体对其特定节肢动物媒介生理的微调适应。 伯氏杆菌基因调控的一个标志是RpoN/rpos调控网络。这种调控级联协调伯氏杆菌成功传播所需的毒力因子的表达。然而，在北美分离的所有回归热螺旋体菌株都不具有功能性的RpoN，这表明RpoN/rpos调节级联在B.hermsii中不起作用。因此，我们最初的重点是开发必要的生化和遗传工具来研究依赖于rpoS、RpoD、BosR和CSRA的基因对已知的赫氏杆菌毒力因子(即VMP和VTP)的调控，特别是在传播周期中从VTP转换到VTP的调控。 在2022财年，我们的研究小组在改进研究赫氏杆菌所需的遗传工具方面取得了重大进展。在过去的五年中，一些报道表明，可变壁虱蛋白(VTP)的表达是成功传播Hermsi复发性热(RF)螺旋体所必需的，并且VTP的上调发生在开始摄食之前的扁虱唾液腺中。这表明，静止唾液腺的条件诱导这一重要蛋白在摄食前表达，以促进快速和成功地传播到新的哺乳动物宿主。我们的初步数据表明，ROS在静息的扁虱唾液腺中存在显著水平。值得注意的是，在体外，ROS(如过氧化氢或过氧化叔丁基)可以诱导包括VTP在内的许多重要蛋白质的表达。利用转录组和蛋白质组学的方法，我们能够确定赫氏芽孢杆菌对ROS抗性的重要因素。 虽然BosR在调节VTP中的作用尚不清楚，但我们已经确定了一种控制VMP-VTP开关的机制，该开关是宿主和媒介获取、定殖和维持所需的。转录后调节因子CSRA在B.hermsii中是必需的，并可能在VMP-VTP开关的调节中发挥作用。为了研究这一重要调控因子的本质，开发了一个可诱导的CSRA突变体，允许在实验中操纵B.hermsii中的CSRA水平。据我们所知，这是在赫氏假单胞菌中产生诱变突变体的第一次报道。此外，还利用体外RNA结合试验筛选了相关RNA转录本上的CSRA结合位点，包括与碳代谢和毒力相关的基因。这些系统和方法的发展对于分析成功传播所需的赫氏杆菌调控网络至关重要。 最后，我们正在研究营养利用在赫氏芽孢杆菌感染周期中的生长和存活中的作用。具体地说，我们正在研究ADS在哺乳动物感染期间对赫氏杆菌生长和增殖的作用。在此之前，我们已经展示了ADS在调节伯氏杆菌的酸胁迫反应中的作用。为了了解Ads在Hermsii感染循环中的作用，我们在精氨酸脱亚胺酶和氨基甲酸激酶方面进行了突变。有趣的是，我们发现赫氏杆菌和伯氏杆菌对ADS的调节不同，而且赫氏杆菌很容易降低感染小鼠体内的精氨酸浓度。初步结果表明，在第一个螺旋体感染高峰期间，Hermsii对精氨酸的利用导致细菌负荷显著增加。此外，Hermsii对精氨酸的消耗也引起了广泛的免疫学变化，目前正在进行研究。

This project is investigating the regulation of key virulence factors in Borrelia hermsii that are required for successful transmission from its tick vector, Ornithodoros hermsi, to a mammalian host. While B. hermsii has the genes encoding similar regulatory proteins as B. burgdorferi (i.e., RpoS, RpoD and the Borrelia oxidative stress regulator, BosR), the responses of the bacterium to environmental signals, such as temperature, reactive oxygen species (ROS), reactive nitrogen species (RNS), dissolved oxygen and pH, are dramatically different. These differences reflect a finely tuned adaptation by these pathogenic spirochetes to the physiology of their particular arthropod vectors. A hallmark of gene regulation in B. burgdorferi is the RpoN/RpoS regulatory network. This regulatory cascade coordinates the expression of virulence factors required for the successful transmission of B. burgdorferi. However, all strains of relapsing fever spirochetes isolated in North America do not harbor a functional RpoN, suggesting that the RpoN/RpoS regulatory cascade does not function in B. hermsii. Therefore, our initial focus has been on developing the necessary biochemical and genetic tools to study the RpoS-, RpoD-, BosR- and CsrA-dependent gene regulation of known virulence factors (i.e., Vmp and Vtp) in B. hermsii with a particular emphasis on the regulation of switching from Vtp to Vmp during the transmission cycle. In FY2022, our research group made significant progress improving the genetic tools necessary for studying B. hermsii. Several reports over the past five years have shown that the expression of the Variable tick protein (Vtp) is required for successful transmission of relapsing fever (RF) spirochetes from O. hermsi and that up-regulation of Vtp occurs in the salivary glands of ticks before the initiation of feeding. This suggests that conditions in the resting salivary glands induce the expression of this important protein prior to feeding in order to promote rapid and successful transmission to a new mammalian host. Our preliminary data suggests that ROS is present at significant levels in resting tick salivary glands. Significantly, ROS (e.g., hydrogen peroxide or t-butyl peroxide) can induce the expression of numerous important proteins, including Vtp, in vitro. Using a transcriptomic and proteomic approach, we were able to identify factors important for B. hermsii resistance to ROS. Although the role of BosR in regulating Vtp remains undefined, we identified one mechanism of control for the Vmp-Vtp switching required for host and vector acquisition, colonization, and maintenance. The post-transcriptional regulator, CsrA, was shown to be essential in B. hermsii and may play a role in the regulation of Vmp-Vtp switching. To study the essential nature of this important regulator, an inducible csrA mutant was developed allowing for the experimental manipulation of CsrA levels within B. hermsii. To our knowledge, this is the first report of the generation of an inducible mutant in B. hermsii. In addition, in vitro RNA binding assays were employed to screen putative CsrA binding sites on RNA transcripts of interest including genes involved in carbon metabolism and virulence. The development of these systems and methods are pivotal for analyzing the regulatory networks in B. hermsii that are required for successful transmission. Finally, we are investigating the role of nutrient utilization in the growth and survival of B. hermsii during its infectious cycle. Specifically, we are investigating the role of the ADS in the growth and proliferation of B. hermsii during mammalian infection. Previously, we have shown a role for the ADS in modulating the acid stress response in B. burgdorferi. To understand the role of the ADS in B. hermsii infectious cycle, mutants were made in the enzymes, arginine deiminase and carbamate kinase. Interestingly, we have discovered that the regulation of the ADS in B. hermsii differs from B. burgdorferi and that B. hermsii readily reduces the concentration of arginine in an infected mouse. Preliminary results show that the utilization of arginine by B.hermsii leads to significantly increased bacterial loads during the first spirochetemic peak. In addition, depletion of arginine by B. hermsii also caused broad immunological changes that are currently under investigation.