Uncovering the Redox Potential of the Global Gene Regulatory Protein DksA in Borrelia burgdorferi

Abstract

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common vector-borne illness in North America. The enzootic cycle of B. burgdorferi involves transmission between Ixodes species tick vectors and vertebrate hosts. This infectious cycle requires the spirochetal bacteria to undergo dramatic shifts in gene expression in order to survive and remain virulent. Currently, it is unclear how B. burgdorferi senses the relevant environmental changes encountered that allow it to regulate these shifts in gene expression. One critical component is the global gene regulatory protein DksA, which is responsible for reducing the expression of housekeeping genes during the stringent response, while promoting the expression of virulence genes during infection. The mechanistic details of how DksA is able to sense environmental signals in B. burgdorferi is poorly understood. DksA harbors a conserved zinc finger motif, coordinated by four cysteine residues, that may serve as a redox sensor of tick-borne reactive nitrogen species (RNS) and reactive oxygen species (ROS). These post-translational redox modifications would enable DksA to direct gene expression required for the completion of the enzootic cycle. The central hypothesis for this study is that the global gene regulatory protein DksA of B. burgdorferi is labile to post-translational modifications to cysteines that coordinate its zinc finger motif caused by ROS and RNS encountered throughout the enzootic cycle, subsequently affecting gene expression. This hypothesis was tested in five ways: 1) complementing a ∆dksA Escherichia coli strain in trans with a library of plasmids encoding B. burgdorferi dksA alleles with cysteine to serine substitutions, 2) developing in cis complemented strains of B. burgdorferi encoding dksA alleles with cysteine to serine substitutions, 3) measuring zinc release from recombinant DksA protein following treatment with ROS or RNS, 4) optimizing the B. burgdorferi in vitro transcription assay to study purified recombinant DksA protein function, and 5) comparing gene expression in a library of B. burgdorferi strains in response to ROS or RNS treatment. The results supported the hypothesis by showing that the cysteine residues of the zinc finger motif are required for B. burgdorferi DksA function within the model organism, the cysteine residues of the zinc finger motif of B. burgdorferi DksA are labile to post-translational modifications by ROS and RNS, and DksA plays a role in the regulation of ROS and RNS induced genes within B. burgdorferi.