Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline

Abstract

SummaryThe Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.

Application of Long Read Sequencing to Determine Expressed Antigen Diversity inTrypanosoma BruceiInfections

“…Despite the limitations of the study design, where we have analysed batches of mice at four time points rather than longitudinal surveys of individual mice, our data across 20 mice and four time points are very consistent with a highly reproducible of pattern of VSG expression over time ( Figure 3 & Table 2). There was a remarkable degree of consistency in identity of dominant VSGs across independent infectionsparticularly as the inoculum used was not a single cell or a cloned inoculum (this is very distinct from, for example, Borrelia, where Pacbio analysis has indicated very little overlap in expressed antigen diversity across replicates from the same starting inoculum [34]). The data demonstrated a consistent emergence of the two sequentially dominant variants at the beginning and end of the infection period (Tb08.27P2.380 and Tb09.v4.0077), although during the period in between the dominant VSGs there was significant diversity in expressed VSGs that was consistent with an inherent degree of stochasticity in the system.…”

Changing of the guard: How the Lyme disease spirochete subverts the host immune response

“…As first described by Zhang et al (35), the vlsE gene in strain B31 is a mosaic of pieces derived from the 15 silent cassettes. The random mixing of genetic information from the silent cassettes into the expression locus gives rise to a theoretical possibility exceeding 10 40 distinct vlsE sequence variants (46). In addition, vlsE variability can also occur through nontemplated sequence changes, as will be discussed below.…”

A Borrelia burgdorferi mini‐vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat

“…In our previous studies on SNP usage during switching at vlsE in the wild-type vls locus, we noted that SNPs from all silent cassettes were transferred to vlsE in the population of analyzed switch variants at approximately the frequency of their occurrence in the silent cassettes (Verhey et al, 2018a). To further characterize switching in the mini-vls system, we analyzed SNP usage from cassette 2 (Fig.…”

“…We used a previously reported software pipeline to process the sequence data (Verhey et al, 2018a) and analyzed switching activity using the Variable Antigen Sequence Tracer (Verhey et al, 2018b).…”