Fig 1.
Schematic illustration of the antigenic variation mechanism at the vls locus of B. burgdorferi B31.
(A) Critical stages of antigenic variation. When mammalian hosts are infected by Ixodes spp. ticks (i), the antigenic variation process is initiated (ii). During this process, the outer surface protein, VlsE, undergoes sequence changes (iii), consequently the variant VlsE would not bind effectively with preformed anti-VlsE antibodies (iv) and thereby enabling the spirochete to establish persistent infection within the host (v). Naïve ticks then acquire the spirochetes from these infected hosts (vi) and are capable of successfully transmitting to subsequent hosts (vii). (B) Molecular mechanism of antigenic variation. The antigenic variation in B. burgdorferi occurs at the vls locus. The vlsE expression site, along with its promoter (P), is located 82 bp from the covalently closed right end of the linear plasmid lp28-1 (telomere, purple). 15 silent cassettes are arranged in an array, oriented oppositely to the vlsE gene. Unknown factor/s within the host trigger a unidirectional, non-reciprocal transfer of genetic information from the silent cassettes to vlsE resulting in production of a composite vlsE that escapes recognition by the humoral immune response of the host. (C) Primary structure of VlsE. Constant regions (CR, pink) on both- N- and C- terminal ends surround the central cassette region demarcated by 17 bp direct repeats (DR1 and DR2, magenta). The central cassette region is further divided into six invariable regions (IR1-6, blue) and six variable regions (VR1-6, yellow). All the recombination events during mammalian infection occurs in VRs. The intergenic region between vlsE and the first silent cassette, 2 (green), contains a 51 bp perfect inverted repeat (shown by bidirectional arrow) that partially overlaps with the promoter (P). The signal sequence (SS) of VlsE is shown in orange. The figure was created with BioRender.com.
Fig 2.
Gene conversion of vlsE can occur in trans.
Schematic representation of (i) circular (pBSV2::vlsE) and (ii) linear vlsE (pBSV2rtel::vlsE) plasmid constructs. vlsE along with its native promoter (P) (yellow) was cloned into the B. burgdorferi- E. coli shuttle vector pBSV2 containing a kanamycin resistance marker (red) and the pncA gene (blue). Plasmid pBSV2::vlsE retained its circular form when transformed into B. burgdorferi 5A10 cells. To linearize the plasmid following transformation into B. burgdorferi, a replicated telomere (rtel, purple) was cloned into the construct, creating pBSV2rtel::vlsE having single telomere at each end. Silent cassettes present on lp28-1 in 5A10 serve as ‘donor’ sequences for gene conversion. The figure was created with BioRender.com.
Table 1.
Results demonstrating the difference in the ability of vlsE copy harbored on a circular or linear plasmid shuttle vector in B31-5A10 strain to persistently infect mice and undergo vlsE gene conversion.
Fig 3.
Construction and analysis of mutations in direct repeats (DR) regions of vlsE.
(A) Schematic drawing of mutations generated in direct repeat (DR) regions (magenta) flanking the central cassette of vlsE (yellow) on the linear shuttle vector pBSV2rtel::vlsE. A stretch of guanine bases in the wild-type sequence (boxed) were replaced with adenine and thymine bases (underlined) in the mutant sequence in both DR1 and DR2 regions as illustrated in the magnified box. Constant regions (CR, pink) are marked on both sides of the central cassette. Plasmids carrying either wild-type or the mutated copy of vlsE were each transformed into B. burgdorferi A1 and 5A10 strains. (B) qRT-PCR assessment of changes in the transcription level of vlsE. Total RNA was extracted from in vitro grown wild-type A1 strain and A1 harboring either wild type or mutated vlsE copy on a linear plasmid, converted to cDNA, and evaluated through ddPCR wherein vlsE transcripts levels were normalized to 1000 copies of flaB using a TaqMan assay. Values were expressed as an average of triplicates ± standard error mean. No significant (ns) difference in the vlsE transcript levels were observed between wild type and DR mutant strains using Student’s paired t-test (p>0.05). The figure was created with BioRender.com.
Fig 4.
Schematic representation of the experimental strategy adopted to assay for vlsE recombination during Bb infection of mice. (i) Shuttle plasmids containing either the wild-type or mutated copy of vlsE were constructed, (ii) B. burgdorferi 5A10 strain was transformed, (iii) groups of three C3H/HeN mice each were infected subcutaneously via needle inoculation, (iv) progress of infection was monitored for 2 weeks by culturing blood at week 1, followed by (v) euthanasia of mice at week 2, (vi) portions of skin, bladder, heart and joint were harvested and cultured in BSK-II media. After a week (vii) total DNA was isolated from the recovered spirochetes and subjected to (viii) PCR-RFLP assay and (ix) DNA sequencing of the variable region of vlsE gene to assess vlsE recombination. The figure was created with BioRender.com.
Table 2.
Effect of DR mutations present in B31-5A10 strain on B. burgdorferi infection in C3H/HeN mice.
Fig 5.
Mutations in direct repeat (DR) regions inhibit in trans gene conversion.
Three groups of C3H mice (3 animals/group) were infected via needle inoculation with Bb 5A10 strain harboring either the wild type or DR mutant copy of vlsE residing on the linear shuttle plasmid pBSV2rtel::vlsE. Wild type B31- A3 was also used to infect mice as a control. Total DNA was extracted from the recovered spirochetes from the bladder tissue at week 2 p.i. and subjected to field-inversion gel electrophoresis. The shuttle vectors harboring vlsE copy (8.6 kb) were gel excised and used as template for amplification of the central cassette region of vlsE. For B31-A3 control mice, the vlsE variable cassette was amplified directly from total DNA containing native lp28-1 plasmid. (A) PCR-RFLP analysis. The amplified vlsE genes were analyzed undigested or digested with HphI both in vitro and after inoculation in C3H mice as indicated. Results showed that both WT controls—(i) A3 and (ii) WT vlsE copy on SV displayed a banding pattern due to introduction of additional and/or removal of HphI sites indicative of vlsE gene conversion while (iii) DR mutant vlsE copy on SV did not show any recombination. SV, shuttle vector; 1, 2, 3 corresponds to mouse 1, mouse 2 and mouse 3 respectively; M, 100 bp marker. (B) Multiple sequence alignment of WT copy of vlsE on the linear shuttle vector. DNA sequencing was performed on PCR product amplified from the wild type vlsE copy of the linear shuttle vector, as described in (A). These amplicons were TOPO cloned and sequenced using M13 primers. The alignment results revealed changes across the six variable regions (VR1-6) of vlsE, indicating recombination events in spirochete populations from all three mice. (C) Multiple sequence alignment of vlsE copy on SV in the DR mutant group. Limiting dilution plating of the spirochetes recovered from each mouse was performed and 15 clones (five from each mouse) were randomly picked for sequencing and processed as above. All the 15 clones showed the mutant sequence in both DR1 and DR2 regions, highlighted in magnified boxes while there were no changes in any of the variable regions implying that the DR mutations in SV inhibit gene conversion in trans. Positions where the sequences match the anchor sequence (vlsE) are colored in pink (and as grey dots in magnified box), while positions that contain mismatches are colored in red. Gaps are indicated by a pink line in white space while insertions relative to the anchor sequence are indicated by a blue bracket. Mutant sequences in DR1 and DR2 are shown in magenta, and the wild type sequence is shown in black in the magnified box. The alignment ruler spans the positions 1–660, starting at the vlsE sequence where primer P243 binds the N-terminal constant region and ending at 16 bp of the C-terminal constant region. To highlight the specific regions where changes occurred during recombination events, a color-coded bar was added: magenta for direct repeat regions (DR1, DR2), blue for invariable regions (IR1-6), and yellow for variable regions (VR1-6). Numbers on the left are the clones recovered from the mice and used in sequencing.
Fig 6.
Introduction of mutations into native vlsE on lp28-1.
(A) PCR-RFLP analysis of native vlsE on lp28-1. The experiment was conducted as outlined in Fig 5. Native lp28-1 (28 kb) was gel excised and used as the template for PCR followed by RFLP assay. Both the (i) WT and (ii) DR mutant exhibited banding patterns that suggests vlsE gene conversion, indicative of recombination events. vlsE amplicon from in vitro grown culture was used as a negative control as before.1, 2, 3 corresponds to mouse 1, mouse 2 and mouse 3 respectively; M, 100 bp marker. (B) Multiple sequence alignment of native vlsE on lp28-1 in DR mutant. 15 clones from three mice were randomly selected and sequenced as described above. One clone showed incorporation of mutations into the native vlsE on lp28-1 in both DR regions (†) from the shuttle vector copy while several more clones were found to incorporate mutations either in the DR1 (green arrow) or DR2 (*) region only. Magnified boxes indicate detailed sequence in DR1 and DR2 regions in all clones. Color coding is same as described in Fig 5. (C) A schematic illustration depicting the transfer or “movement” of mutations within DR1 and DR2 regions from the vlsE gene copy carried on the shuttle vector (pBSV2rtel::vlsE:DR) to the vlsE gene copy residing on the native lp28-1 plasmid in the 5A10 strain of B. burgdorferi. The recipient vlsE gene copy on lp28-1 is marked with asterisks for clarity.
Table 3.
Summary of DR mutations and gene conversion association in B31-5A10 strain.
Fig 7.
Introduction of mutations in invariable regions (IR) of the central cassette of vlsE.
(A) A schematic representation of mutations generated in invariable regions of the central cassette of vlsE on the linear shuttle vector pBSV2rtel::vlsE. Alanine coded by 5’ GCT 3’ at positions 220 in IR4 (green) and 282 in IR6 (orange) in the wild type sequence (boxed) was changed to 5’ TAA 3’ coding for stop codon in the mutant sequence (underlined). CR, constant regions (pink); IR, invariable region (blue); DR direct repeat (magenta). (B) Multiple sequence alignment of vlsE copy on SV in A220*. PCR products from the mutant group were TOPO cloned and sequenced using M13 primers as before. All three clones retained the stop codon sequence in IR4 (green). (C) Multiple sequence alignment of the native vlsE copy on lp28-1 in A220*. One out of three clones sequenced for native vlsE in the A220* mutant group incorporated the stop codon (green). (D) Multiple sequence alignment of vlsE copy on SV in A282*. All three clones retained stop codon sequence in IR6 (orange). (E) Multiple sequence alignment of native vlsE copy on lp28-1 in A282*. Sequencing of vlsE amplified from native lp28-1 from IR6 mutant group showed incorporation of stop codons from SV in two clones. Color coding is same as described in Fig 5. Subpart (A) was created with BioRender.com.
Table 4.
Culture data showing the persistence of infection in C3H/HeN mice harboring stop codons in vlsE copy on shuttle plasmid in B31-5A10 strain.