Role of the Hypothetical Protein BB0563 During Borrelia Burgdorferi Infection in Animals

The alternative sigma factor RpoS in Borrelia burgdorferi, the etiological agent of Lyme disease, has long been postulated to regulate virulence-associated genes other than and . Here, we demonstrate that , a gene encoding a hypothetical protein, is regulated by RpoS and contributes to the optimal infectivity of B. burgdorferi. When B. burgdorferi was exposed to environmental stimuli, showed similar expression patterns as , , and . Expression of was significantly downregulated when was inactivated and was restored in the complemented strain. By using rapid amplification of cDNA ends (RACE) and luciferase reporter assays, a functional promoter was identified in the regulatory region upstream of . Gene expression from this promoter was drastically decreased in the mutant. We next investigated the role of during animal infection. By using quantitative reverse transcription-PCR (RT-PCR), we found that was highly expressed in mouse tissues during infection. We further created a -deficient mutant in a bioluminescent B. burgdorferi strain and examined infection dynamics using imaging. Relative to the parental and complemented strains, the mutant showed a delayed infection pattern and bacterial load was reduced. Another deletion mutant was also created in the strain 297 background, and quantitative PCR (qPCR) analysis revealed a significantly lower spirochetal burden in tissue samples collected from animals infected with the mutant. In addition, localization studies indicate that BB0563 is not exposed on the cell surface but is associated with outer membrane. Taken together, these results suggest that is required for optimal infectivity of B. burgdorferi during experimental infection.