Poster The 5th Prato Conference on Pore Forming Proteins 2021

The role of P66 porin function in infectivity of the Lyme disease spirochete Borrelia burgdorferi (#100)

Christa H Fierros 1 , Michael Curtis 2 , Julie Kessler 1 , Beth Hahn 1 , Marine Vandewelle-Capo 3 , Mari Bonde 3 , Sven Bergstrom 3 , Jenifer Coburn 1
  1. Medical College of Wisconsin, Milwaukee, WI, United States
  2. Pediatrics, Baylor College of Medicine, Houston, Texas, United States
  3. Molecular Biology, Umea University, Umea, Sweden

Lyme disease, which results from infection by the spirochetal bacterium 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. To understand the in vivo significance of P66 porin function to Bb infectivity, we have created P66 mutants by site-directed mutagenesis (SDM), developed an antibiotic-based screen for P66 porin function, and are infecting mice with the P66 porin mutants.

We determined that treatment of WT Bb with low levels of vancomycin causes spontaneous mutations in p66. Most mutants have P66 nonsense mutations, but some have missense mutations. These mutants have altered porin function as determined by our antibiotic-based screening method. This method is based on liquid culture of strains +/- 1µg/ml vancomycin and monitoring culture density by darkfield microscopy for 3 days. WT is sensitive to vancomycin while Δp66 Bb strains and the spontaneous mutants are resistant. This method was verified by black lipid bilayer. We re-created the missense mutations by SDM for the purpose of mouse infections to determine effects on overall infectivity and tissue burdens through ID50 experiments and qPCR from infected tissues. Although an ongoing process, these mutants are infectious in mice and retain the missense mutation. Tissue burdens have yet to be quantified. Thus it appears that Bb can tolerate some alterations in P66 porin function during mammalian infection.

Understanding the porin properties of P66 will guide us to understanding the in vivo significance of this important protein in Bb infection.

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