Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion

Abstract

The Lyme disease spirochete Borrelia burgdorferi possesses 15 silent vls cassettes and a vls expression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5' and 3' coding sequences of vlsE that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent vls cassettes recombine in the vlsE cassette region through a unidirectional process such that the sequence and organization of the silent vls loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.

FIG. 1

Derivation of B. burgdorferi B31 clones in C3H/HeN mice. Clone B31-5A3 was the parental strain used in this study. Clones M1e4C and M1e4A were cultured from a single ear biopsy from a C3H/HeN mouse at 28 days postinfection with B31-5A3 (41). Similarly, clone 1396D was derived from a skin biopsy of a mouse inoculated with 10⁵ M1e4C 28 days previously.

FIG. 2

Nucleotide sequence comparison between the parental vlsE allele (vlsE1) and two derivative alleles, m1e4A and m1e4C. Identical nucleotides and gaps are marked as dashes and dots, respectively; nonidentical nucleotides are shown as letters. The 17-bp direct repeats flanking the vlsE cassette region are shaded. The predicted ribosome binding site (RBS), putative start codon (M), and putative stop codon (∗) are indicated. Locations and directions of oligonucleotides used in this study are marked with arrows.

FIG. 3

Detection of the vlsE noncassette sequences. Southern blots of restriction enzyme-treated plasmid DNA from the parental strain B31-5A3 and two progeny strains M1e4A and M1e4C were hybridized with oligonucleotide probe R4290, representing the 3′ noncassette region of vlsE. The molecular sizes of DNA bands are indicated in kilobases on the left side.

FIG. 4

Evidence for unidirectional recombination of silent vls cassette segments into vlsE. (A) Diagram of locations of oligonucleotide probes in vlsE and the silent vls cassettes in the parental strain B31-5A3 and its derivative strains M1e4A and M1e4C. (B) Hybridization of oligonucleotide probe R4232 with the blots of restriction enzyme-digested plasmid DNA preparations from B31-5A3, M1e4A, and M1e4C. In all lanes, the probe hybridized to a single band corresponding to a region containing the silent cassette vls11 in both strains B31-5A3 and M1e4A. However, for strain M1e4C, two bands were detected in each digest that corresponded to the vlsE region with the second band (arrows). The molecular sizes of DNA bands are indicated in kilobases on the left side.

FIG. 5

Loss of the previously expressed vls cassette segments. The total DNA blot of M1e4C-derived clone 1396D was hybridized with oligonucleotide probe R4232. In all lanes, the probe hybridized to a single band containing the silent cassette vls11. The molecular sizes of DNA bands are indicated in kilobases on the left side.