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

National Institute Of Allergy And Infectious Diseases

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Abstract

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.