Understanding The Mechanism and Extent of Vlse Recombination In Borrelia Burgdorferi Using Next-Generation Sequencing

<p><em>B.</em> <em>burgdorferi</em>, the causative agent of Lyme disease, have an elaborate antigenic variation system that involves varying the sequence of <em>vlsE. </em>Previous studies have shown that <em>vlsE</em> antigenic variation occurs continuously inside mammalian hosts. Variation has not been shown previously to occur in<em> in vitro</em> or in ticks. We hypothesized that the induction of <em>vlsE</em> 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 <em>vlsE </em>recombination within mouse tissues and <em>in vitro</em> model systems. Using these approaches, we were able to detect<em> vlsE</em> variants in axenic cultures of <em>B. burgdorferi</em> 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<em> in vitro </em>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 <em>in vitro</em> cultures. Thus, tissue explant co-cultures do not seem to promote a higher recombination rate than in <em>in vitro</em> axenic culture. Moreover, high-throughput PacBio sequencing was found to be an effective means of analyzing single molecule sequencing variation in the robust <em>vlsE </em>antigenic variation system.</p>

Name thesis:degree_name

Masters of Science (MS)

Level thesis:degree_level

Thesis (MS)

Year dc:date.available

2016

Author dc:creator

• Tyagi, Surabhi

Contributors dc:contributor

• Steven Norris
• David Volk
• Cesas Arias

• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/702

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1746