Lyme disease, which results from infection by Borrelia burgdorferi (Bb) via the bite of an infected Ixodes tick, is the most prevalent arthropod-borne disease in the United states and Europe. Untreated Lyme disease can lead to carditis, neuritis, arthritis, and other serious symptoms. Previous work has shown that P66, a porin and adhesin, is upregulated during murine infection and is essential for infectivity, but adhesin-deficient P66 mutants remain infectious. We hypothesize that the porin function of P66 plays a role in infectivity. We aim to use in vitro and in vivo techniques to identify substrates with P66-dependent entry, the crucial porin residues, and ultimately the in vivo significance of P66 to Bb infectivity.
To gauge the size of substrates with P66-dependent entry, we utilized liquid culture of WT and Δp66 Bb strains in BSKII medium supplemented with periplasm-active antibiotics of varying molecular weights and measured the response in culture density over time. Vancomycin entry is P66-dependent and removing vancomycin from the media of vancomycin-resistant clones does not restore P66 production. We have developed the use of vancomycin susceptibility as a screen for P66 function in all of our mutants; this is being confirmed by black lipid bilayer assay. In testing additional antibiotics, we have observed that the periplasmic entry of lower weight molecular antibiotics appears independent of P66 while higher molecular weight antibiotics may be P66 dependent. Spontaneous vancomycin resistant clones exhibit a loss of P66 expression as determined by western blotting. We are currently characterizing the antibiotic resistance mechanism by sequencing and qRT-PCR. In parallel, we are generating targeted porin mutants, which will be characterized for porin function and their effects on murine infectivity.
Understanding the porin properties of P66 will guide us to understanding the in vivo significance of this important protein in Bb infection.