Site-specific alterations of key residues of a transcriptional regulator impact pathophysiology of Borrelia burgdorferi

Lyme Disease is a prevalent tick-borne illness in the U.S. caused by the bacterium Borrelia burgdorferi (Bb) and is transmitted through the Ixodes scapularis tick. BosR (Borrelia oxidative stress Regulator), a major regulator of gene expression, is a metal-dependent DNA-binding protein. BosR shares significant homology with PerR, a protein present in Bacillus subtilis, both of which are involved in modulating response to oxidative stress. Additionally, BosR functions as a homodimer, containing a DNA-binding domain and has been shown to bind upstream regions of rpoS – the major sigma factor regulating the transcriptional profile of Bb upon exposure to the incoming blood meal during transition from the tick vector to vertebrate host. While the role of a key residue (Arginine at position 39) in BosR has been shown to alter its binding to select upstream regions of genes, the role of two redox-sensor CXXC motifs at its C-terminus and the two Histidines predicted to bind to Zn2+ remains undetermined. To address these gaps in knowledge, four site-specific mutants were generated that included two zinc-binding residues at position 37 and 111 along with two CXXC motifs, which disrupt the disulfide bond formation necessary for dimerization of BosR. We first sought to determine whether the overall phenotypic outcome in terms of RpoS-dependent gene expression was affected, as well as binding capabilities to certain regulatory regions. The mutants did not make OspC, indicating the residues play a critical role in functional activity of BosR to activate RpoS and rpoS-dependent regulon. We also determined whether the aforementioned mutants interfere with dimerization capabilities of BosR, which is required for DNA-binding, through the use of a bacterial two-hybrid system. The role of these site-specific mutants in regulating the oxidative stress response in Bb and the ability to colonize the mouse model of Lyme disease will lead to a better understanding of the role of BosR. Molecular characterization of the role of BosR will unravel novel mechanisms to disrupt survival of Bb during the mammalian and tick phases of its natural life cycle.