REGULATORY ROLES OF THE CONSERVED NUCLEIC ACID–BINDING PROTEIN SPOVG IN BORRELIA BURGDORFERI

Abstract

Understanding how pathogens rapidly adapt to changing host environments is essential for developing strategies to combat infectious diseases. Lyme disease, caused by the spirochete Borrelia burgdorferi, affects approximately 476,000 individuals annually in the United States. Transmitted by Ixodes ticks, B. burgdorferi causes multisystem disorders with highly debilitating complications. Despite its prevalence, no human vaccine is currently available, and delayed diagnosis often leads to treatment failure or persistent symptoms. The spirochete’s ability to persist across distinct host niches relies on sophisticated gene regulation, yet the molecular mechanisms of this host adaptation remain incompletely understood. One key regulatory factor of bacterial adaptation is SpoVG, a conserved protein found across Eubacteria including spirochetes. Originally linked to sporulation in Bacillus spp., SpoVG in B. burgdorferi appears to regulate gene expression as both a repressor and activator. To delineate the role of SpoVG in B. burgdorferi spoVG deletion mutants were generated in the virulent B. burgdorferi B31-A3 background. The loss of SpoVG resulted in increased protein levels of some virulence factors, such as BosR, RpoS, OspC, BBK32, and DbpA, accompanied by a reduction in OspA, indicating that SpoVG broadly modulates the B. burgdorferi essential virulence regulon. Furthermore, global transcript dysregulation was observed. RNA sequencing identified over 200 statistically significant differentially expressed genes in the ΔspoVG mutant. Complementary to this, quantitative RT-PCR demonstrated significant upregulation of bosR, rpoS, ospC, bbk32, and dbpA, and downregulation of ospA in the absence of SpoVG. Biochemical analyses were performed using recombinant SpoVG to determine whether SpoVG directly regulates gene expression by binding gene promoters. Electrophoretic mobility shift assays and surface plasmon resonance revealed that SpoVG poorly binds the rpoS promoter but specifically binds the bbk32 promoter. BBK32 is a multifunctional surface lipoprotein that promotes mammalian infection by binding host fibronectin and inhibiting complement activation. Binding was mapped to a discrete region containing palindromic TAATTT motifs and required the conserved arginine residues Arg53 and Arg54 among Eubacterial SpoVG homologs, underscoring their essential role in sequence-specific DNA binding. Interestingly, the TAATTT motif has been shown to be critical for the DNA-binding activity of SpoVG in Staphylococcus aureus and is present in the known SpoVG target in the glpFKD promoter that is responsible for the uptake and utilization of glycerol. Collectively, these findings demonstrate that SpoVG functions as a regulatory protein in B. burgdorferi that directly represses bbk32 transcription through sequence-specific DNA binding while influencing the BosR–RpoS axis, a central pathway controlling virulence gene expression during mammalian infection. This work expands understanding of gene control mechanisms that govern B. burgdorferi virulence and identifies SpoVG as a modulator of Lyme disease pathogenesis.