Date of Graduation
8-2016
Document Type
Thesis (MS)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Masters of Science (MS)
Advisor/Committee Chair
Steven Norris
Committee Member
David Volk
Committee Member
Cesas Arias
Committee Member
Kevin Morano
Committee Member
Nayun Kim
Abstract
B. burgdorferi, the causative agent of Lyme disease, have an elaborate antigenic variation system that involves varying the sequence of vlsE. Previous studies have shown that vlsE antigenic variation occurs continuously inside mammalian hosts. Variation has not been shown previously to occur in in vitro or in ticks. We hypothesized that the induction of vlsE recombination requires contact with dense arrays of host tissue cells and/or ECM components. To test this hypothesis, two methods, quantitative PCR and high-throughput sequencing were used determine the extent and nature of vlsE recombination within mouse tissues and in vitro model systems. Using these approaches, we were able to detect vlsE variants in axenic cultures of B. burgdorferi as well as co-cultures with mouse skin and heart tissues; these results were compared with those from mice infected for 7 days. Analysis of PacBio single molecule real-time (SMRT) sequencing indicated the presence of 0.84% to 1.18% variants in pure in vitro cultures and 0.79% to 1.22% in tissue explants, as compared 36% to 57% for organisms from mouse bladder tissue 7 days post inoculation. Statistical evaluation of the variants showed that the rate of recombination in tissue explants was not significantly different from the rate of recombination in in vitro cultures. Thus, tissue explant co-cultures do not seem to promote a higher recombination rate than in in vitro axenic culture. Moreover, high-throughput PacBio sequencing was found to be an effective means of analyzing single molecule sequencing variation in the robust vlsE antigenic variation system.