A Molecular Genetic Investigation of the Regulation of Outer Surface Protein C in Borrelia Burgdorferi: Identification and Characterization of the Novel Regulatory Protein BBD18

Lyme disease, caused by Borrelia burgdorferi, is the most prevalent arthropod-borne disease in the U.S. In nature, B. burgdorferi is maintained in an enzootic cycle between Ixodes ticks and mammalian hosts. In order to cause infection in mammals, B. burgdorferi spirochetes within a feeding tick sense environmental changes and subsequently alter their gene expression and protein profiles. As part of this adaptation, spirochetes within the tick midgut downregulate outer surface protein (Osp) A and upregulate OspC. Although OspC is an essential B. burgdorferi virulence factor needed for early mammalian infection, it is also a target for the mammalian acquired immune response, and thus, OspC is repressed soon after B. burgdorferi establishes infection. It has been shown that the central Rrp2/RpoN/RpoS pathway is essential for the upregulation of OspC and other genes important for mammalian infection. However, many key aspects in the strict regulation of OspC remain undefined. In order to investigate the complex regulatory mechanisms controlling OspC expression, we adapted the lacZ reporter system from Escherichia coli for use in B. burgdorferi. Using this lacZ system and other molecular genetic approaches, we identified BBD18 as a novel factor that has the potential to negatively regulate OspC. Expression of BBD18 in B. burgdorferi repressed transcription of ospC and abrogated infection in mice. We determined that BBD18 likely interfaces with the central Rrp2/RpoN/RpoS pathway and exerts its effect on OspC through the repression of RpoS. Structural modeling indicated that BBD18 has a putative DNA-binding motif, and site-directed mutagenesis within this domain abrogated BBD18\u27s ability to repress OspC and prevent infection of mice. We propose that the BBD18 protein acts in concert with other regulatory factors to precisely control gene regulation in B. burgdorferi during the enzootic cycle