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

Abstract

Lyme disease, also known as Lyme borreliosis, is the most common tick-transmitted disease in the Northern Hemisphere. The disease is caused by the bacterial spirochete Borrelia burgdorferi and other related Borrelia species. One of the many fascinating features of this unique pathogen is an elaborate system for antigenic variation, whereby the sequence of the surface-bound lipoprotein VlsE is continually modified through segmental gene conversion events. This perpetual changing of the guard allows the pathogen to remain one step ahead of the acquired immune response, enabling persistent infection. Accordingly, the vls locus is the most evolutionarily diverse genetic element in Lyme disease-causing borreliae. Small stretches of information are transferred from a series of silent cassettes in the vls locus to generate an expressed mosaic vlsE gene version that contains genetic information from several different silent cassettes, resulting in ∼10⁴⁰ possible vlsE sequences. Yet, despite its extreme evolutionary flexibility, the locus has rigidly conserved structural features. These include a telomeric location of the vlsE gene, an inverse orientation of vlsE and the silent cassettes, the presence of nearly perfect inverted repeats of ∼100 bp near the 5' end of vlsE, and an exceedingly high concentration of G runs in vlsE and the silent cassettes. We discuss the possible roles of these evolutionarily conserved features, highlight recent findings from several studies that have used next-generation DNA sequencing to unravel the switching process, and review advances in the development of a mini-vls system for genetic manipulation of the locus.

Keywords: Borrelia; DNA mismatch repair; DNA recombination; G-quadruplex; Lyme disease; antigenic variation; bacterial genetics; bacterial pathogenesis; gene conversion; genetic polymorphism; infectious disease.

Figure 1.

Cartoon of antigenic variation. Some pathogens will thwart their recognition by the host immune system by continually changing a prominent surface antigen through changes in allele expression or gene conversion events to modify the expressed allele. In the schematic, the changing surface antigen from the surface of a pathogen is depicted by the red and blue ovals.

Figure 2.

Schematic of the vls antigenic variation locus of B. burgdorferi strain B31. A, the vls expression locus (vlsE) with its promoter (P) is located 82 bp from the right covalently closed hairpin end of the linear plasmid lp28-1. To the left of the promoter and intergenic region (gray) are 15 silent cassettes carrying information corresponding to the variable region of vlsE and situated in the opposite orientation. B, the vlsE region is shown in greater detail, with the constant regions (CR) shown in yellow and the variable region, which corresponds to the information carried in the vls cassettes, shown in blue. The variable region is flanked by 17-bp DRs. To the left of vlsE is its promoter, with the −10 and −35 sequences shown as green bars. Also shown by the bidirectional arrow is a 100-bp perfect IR that partially overlaps the promoter. C, an enlargement of the vlsE gene shows the product of multiple recombinational switching events that result in the copying of genetic information from the silent cassettes into the expression locus, producing a mosaic vlsE carrying information from a number of the silent cassettes. D, an inverted repeat found in the promoter region of the vlsE gene is shown in its normal linear configuration and as an extruded cruciform promoted by negative supercoiling or DNA unwinding from replication or transcription. Modified from Ref. . This research was originally published in Molecular Microbiology. Castellanos, M., Verhey, T. B., and Chaconas, G. A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat. Mol. Microbiol. 2018; 109:710–721. © John Wiley & Sons, Inc.

Figure 3.

Distribution of G3⁺-runs on the linear plasmids carrying the vls loci in Borrelia garinii Far04, Borrelia spielmanii A14S, and Borrelia mayonii MN14-1539 and levels of amino acid sequence identity in RecA, OspC, VlsE constant and VlsE variable regions in Lyme borreliae. A–C, G-runs of 3 nucleotides or more were counted in lp28–9 (CP001316.1), lp28–8 (CP001465.1) and lp28–10 (NZ_CP015805.1) in the three above species, respectively. The distribution of G-runs on both strands of non-vls DNA, in the vls silent cassettes and in the vlsE gene are plotted. The number of G-runs in a codon-optimized vlsE gene generated by reverse translation of the amino acid sequence (https://www.bioinformatics.org/sms2/rev_trans.html) using B. burgdorferi B31 codons (https://www.kazusa.or.jp/codon, species ID: 224326) is also shown. D, Sequence alignments were performed for the 25 available full length RecA sequences that were recovered in a BLAST search (see Table S1 for accession numbers) using DNASTAR MegAlign Pro. The percent identity was determined as the mean of the complete set of pairwise alignments and is shown ± the standard deviation. For OspC and the VlsE variable and N-terminal constant regions the same analysis was performed using sequences from a set of 15 Borrelia strains where both OspC and full-length or near full-length VlsE sequences were available in each strain (see Table S1 for accession numbers). (Please note that the JBC is not responsible for the long term archiving and maintenance of this site or any other third pary hosted site.)

Figure 4.

Heat map of mutational frequencies in B31 VlsE and switching at vlsE in SCID versus WT mice. A, variant vlsE sequences were mapped onto the crystal structure of VlsE (47) (Protein Data Bank entry 1L8W) onto both protomers of the dimer structure. The frequency of mutation was depicted using a continuum of color with 0% change indicated by dark blue, ∼37% by green, ∼55% by yellow, and ∼74% by red. For a 360° rotation of the structure, see Video S1. B, the positions of purifying (red) and diversifying (green) mutations are shown on the crystal structure of B31 VlsE. Purifying residues are those with the highest SCID/WT ratios, and diversifying residues are those with the highest WT/SCID ratios. The dotted line is the axis of 180° rotational symmetry. For a 360° rotation of the structure, see Video S2. A and B are from Ref. . This research was originally published in Molecular Microbiology. Verhey, T. B., Castellanos, M., and Chaconas, G. Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline. Mol. Microbiol. 2018; 107:104–115. © John Wiley & Sons, Inc.

Figure 5.

Rate of recombinational switching at vlsE and correlation of cassette usage and intact DR repeats. A, inferred switch event accumulation per read is shown from 0 to 5 weeks post-infection. Linear regression by least squares is shown, with the gray area indicating the region of 95% confidence. The switching rate was measured in SCID mice, where the variants generated are stable and not cleared by immune selection. B, the use of each cassette as a donor of genetic information to vlsE was determined and plotted against the number of completely conserved flanking 17-bp direct repeats. Error bars, S.D. A and B are from Ref. . This research was originally published in Cell Reports. Verhey, T. B., Castellanos, M., and Chaconas, G. Antigenic variation in the Lyme disease spirochete: new insights into the mechanism of recombinational switching with a suggested role for error-prone repair. Cell Reports. 2018; 23:2595–2605. © Elsevier Inc.

Figure 6.

Distance between inferred switch events in spirochetes with two switches at 1 week post-infection and correlation of nontemplated SNPs with the number of switch events. A, the distance between inferred switch events was determined and plotted three ways: using the minimal possible switch lengths (which have the largest distance between switches), the maximal possible switch lengths (which have the shortest distance between the switches), or the midpoint between minimal and maximal. Each value was compared with the distance between 10⁶ randomly generated switch variants. B, the number of nontemplated SNPs per read was enumerated and plotted against the number of templated switch events in the same read (reads with 1–10 switches were analyzed). The least-squares regression line for the data is in red, and the 95% confidence limits are indicated by gray shading. Error bars, S.E.M. A and B are from Ref. . This research was originally published in Cell Reports. Verhey, T. B., Castellanos, M., and Chaconas, G. Antigenic variation in the Lyme disease spirochete: new insights into the mechanism of recombinational switching with a suggested role for error-prone repair. Cell Reports. 2018; 23:2595–2605. © Elsevier Inc.