Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi

Abstract

The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host. vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsE sequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into the vlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

FIG. 1

Overall experimental strategy for examining the kinetics of vlsE variation. (A) The overall structure of the vlsE and silent vls cassette loci in B31-5A3 as previously described (33). (B) Infection of C3H/HeN mice with low-passage B. burgdorferi B31-5A3. Organisms were cultured by using skin biopsies obtained on days 4, 7, 14, 21, and 28 after initial infection. Clonal populations were prepared by subsurface plating and used as a source of DNA templates for PCR amplification and sequence analysis of the vlsE cassette region. DR, 17-bp direct repeats.

FIG. 2

Kinetics of vlsE sequence variation during infection of C3H/HeN mice with B. burgdorferi B31-5A3. PCR products corresponding to the regions shown were amplified from B. burgdorferi clones isolated on days 4, 7, 14, 21, and 28 postinfection in C3H/HeN mice. The predicted amino acid sequences for days 4, 14, and 28 are shown; all nucleotide sequences and predicted amino acid sequences are available through GenBank. The 4-day clones 1360A to 1360G, the 14-day clones 1373A to 1373G, and the 28-day clones 1394B to 1394F were prepared from a single skin biopsy culture from mouse A at each time point. The additional clones shown were isolated from other infected mice in the same experiments. Residues identical to the VlsE cassette region of B31-5A3 (vlsE1) are marked as dashes (−); dissimilar and similar amino acids are shown in uppercase and lowercase, respectively; gaps are indicated by periods. The six variable regions (VR) VR-I through VR-VI are shaded.

FIG. 3

Predicted peptide sequence alignment of the parental B31-5A3 vlsE cassette region (vlsE1) and the derivative alleles of progeny clones obtained 7 and 12 months postinfection. The sequence similarity is depicted as described for Fig. 2.

FIG. 4

Occurrence of vlsE sequence variation in CB-17 SCID mice. The predicted peptide sequence alignment of the parental B31-5A3 allele vlsE1 and derivative vlsE alleles obtained from B. burgdorferi clones isolated on days 4, 14, and 28 postinfection in SCID mice are shown. The 4-day clones 1365A to 1365F, the 14-day clones 1379A to 1379E, and the 28-day clones 1395A to 1395F were each prepared from a single skin biopsy culture from SCID mouse A. The additional clones shown were isolated from other infected mice in the same experiment. The predicted stop codons are marked by asterisks. The sequence similarity is shown as described for Fig. 2.

FIG. 5

Continued variation of vlsE following infection of mice with B. burgdorferi clone M1e4C. The vlsE cassette region predicted amino acid sequence of the parental allele m1e4C was aligned with those of seven derivative alleles obtained from skin biopsies 28 days after infection of C3H/HeN mice. The sequence of VlsE1 from strain B31-5A3 (from which M1e4C was derived) has been included for comparison. The predicted amino acid sequence similarity is shown as described for Fig. 2.