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

Abstract

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.

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

“…It seems unlikely that the inverted repeat serves as a recognition site for a sequence‐specific binding protein because of the sequence divergence between the inverted repeats in B31 and JD1. In contrast, our recent finding that the B31 inverted repeat is essential for antigenic switching at vlsE on a circular mini‐ vls plasmid (Castellanos et al , ) suggests that the inverted repeat may play a role in the recombinational switching process.…”

“…In addition, the range of recombination donor sequences and their properties, and the effect of immune selection at vlsE in B. burgdorferi B31 were also analyzed (Verhey et al , ). We have also applied this sequencing and analysis technology to detect low levels of switching behavior from genetically manipulated vls systems that would have appeared to be inactive using other assays (Castellanos et al , ).…”

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

“…To circumvent the inability to genetically manipulate the 10-kb locus, a mini-vls system was developed by direct cloning into a high-passage, highly transformable B. burgdorferi HB19 strain (93). Constructs unclonable in E. coli were recovered in the high-passage B. burgdorferi recipient strain and subsequently transferred into a low-passage strain for mouse infections and NGS analysis of recombinational switching at vlsE.…”

“…However, the inability to genetically manipulate the locus made it impossible to investigate this issue until now. Development of the mini-vls system on both a circular and a linear form of the same plasmid has allowed examination of this question (93). The finding from NGS-switching analysis was that when the upstream IR was present, switching frequency on the circular and linear plasmids was the same.…”

Recent discoveries and advancements in research on the Lyme disease spirochete Borrelia burgdorferi

“…This work was performed with B. burgdorferi strain B31, and similar results were obtained with strain JD1, which differs in several respects: it lacks 17 base-pair direct repeats in the vls system, its silent cassettes are arranged differently, the inverted repeat sequences at the vls locus are different, and the vls sequences themselves are different 32 . Investigations into other features of the vls locus, such as the long inverted repeat that resides in the vlsE promoter, have been undertaken through the construction of mini- vls plasmids 33 . This work suggests that vlsE switching occurs in cis and that the inverted repeat is not required for switching to occur 33 .…”