Next-Generation Sequencing of vlsE Recombinational Switching in the Lyme Spirochete
Abstract
Borrelia burgdorferi and other spirochetes that cause Lyme disease effectively evade the acquired immune response through antigenic variation. The VlsE antigen is expressed on the spirochete surface during mammalian infection. Virtually unlimited numbers of variants are generated through segmental gene conversion events at the vlsE gene from a series of nearby silent cassette sequences that are homologous to the variable region of vlsE. In contrast to other antigenic variation systems, the molecular mechanism for this switching is unknown. Switching at vlsE is dependent on mammalian host factors and the only known requirement is the RuvAB branch migrase; RecA and many of the other homologous recombination proteins in B. burgdorferi are not required. In this study, we developed a new assay for switching at vlsE based on PacBio sequencing and an analytical pipeline that allows the analysis of tens of thousands of full-length variants. We developed the first fully-automated and unbiased method to accurately identify switch events and non-templated mutations from sequence data. This software also contains a large suite of dataset management and analysis tools to quantify many aspects of switch events and the switching process in the dataset. Following a time-course of B. burgdorferi infection in immunocompetent and immunocompromised mice in a variety of tissues, we uncover a series of new insights into recombinational switching at vlsE. We demonstrate that although switching requires mammalian host factors, the rate of vlsE switching is unaffected by the presence or absence of the required immune system. We identify residues that undergo diversifying and stabilizing selection in the VlsE protein in the presence of acquired immunity. We also report accurate rates of recombination and nontemplated mutation, the size and origin of switch events, a role for local sequence homology in promoting switching, and that switch events accumulate in a clustered rather than uniform fashion. We also quantify a secondary mechanism of sequence variation by demonstrating that polymerase slippage generates in-frame, surface-localized insertions and deletions that contribute to VlsE variability.
Description
Keywords
Bioinformatics, Microbiology, Biology--Molecular
Citation
Verhey, T. (2017). Next-Generation Sequencing of vlsE Recombinational Switching in the Lyme Spirochete (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/27102