Antigenic variation in the Lyme spirochete: detailed functional assessment of recombinational switching at vlsE in the JD1 strain of Borrelia burgdorferi

Abstract

Summary Borrelia burgdorferi is a causative agent of Lyme disease and establishes long‐term infection in mammalian hosts. Persistence is promoted by the VlsE antigenic variation system, which generates combinatorial diversity of VlsE through unidirectional, segmental gene conversion from an array of silent cassettes. Here we explore the variants generated by the vls system of strain JD1, which has divergent sequence and structural elements from the type strain B31, the only B. burgdorferi strain in which recombinational switching at vlsE has been studied in detail. We first completed the sequencing of the vls region in JD1, uncovering a previously unreported 114 bp inverted repeat sequence upstream of vlsE. A five‐week infection of WT and SCID mice was used for PacBio long read sequencing along with our recently developed VAST pipeline to analyze recombinational switching at vlsE from 40,000 sequences comprising 226,000 inferred recombination events. We show that antigenic variation in B31 and JD1 is highly similar, despite the lack of 17 bp direct repeats in JD1, a somewhat different arrangement of the silent cassettes, divergent inverted repeat sequences and general divergence in the vls sequences. We also present data that strongly suggest that dimerization is required for in vivo functionality of VlsE.

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

“…85 and 86. Although switching at the vlsE locus of strain B31 has been known to exist for 2 decades, an in-depth analysis of this phenomenon in other strains has not been undertaken. A recent NGS/VAST analysis of switching at vlsE in B. burgdorferi strain JD1 was also undertaken (49). Despite differences in genetic background, a less structured vls locus, a completely different IR, and the lack of DRs, the properties of switching in this strain were found to be quite similar to those observed for B31.…”

“…They comprise five unique IR sequences ranging in size from 93 to 122 bp and have thus far been found in strains from three Lyme Borrelia species: B. burgdorferi, B. garinii, and B. mayonii. The five unique inverted repeats display an average pairwise identity of 49% with a range of 40 -72% compared with a pairwise identity of 44% for randomly scrambled sequences of the same base composition (49). The five distinct IRs therefore show little relatedness at the sequence level.…”

“…Recently, more IRs have been uncovered by targeted sequencing of several vlsE promoter regions using cycle sequencing, which does not require cloning in E. coli and by the use of next-generation sequencing technology for the second wave of sequencing of Borrelia genomes (see Ref. 49 and Table S1). At this time, the sequence of a total of nine IRs has been documented, including several new sequences reported here ( Fig.…”

“…Analysis of the switching process using long read sequencing and VAST software has revealed a variety of important new information (46,49,85). Analyses were performed using B. burgdorferi B31 to infect both WT and SCID (severe combined immunodeficient) mice.…”

“…An analysis of residues undergoing purifying versus diversifying selection was performed by comparing the frequency of mutations at each position in VlsE in WT versus SCID (no immune selection) mice (46,49). Purifying selection results in maintenance or enrichment of amino acids at given positions, whereas diversifying selection results in diversification of the amino acids at given positions.…”