Variable VlsE is critical for host reinfection by the Lyme disease spirochete

Abstract

Many pathogens make use of antigenic variation as a way to evade the host immune response. A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is antigenic variation of the VlsE surface protein. Recombination results in changes in the VlsE surface protein that prevent recognition by VlsE-specific antibodies in the infected host. Despite the presence of a substantial number of additional proteins residing on the bacterial surface, VlsE is the only known antigen that exhibits ongoing variation of its surface epitopes. This suggests that B. burgdorferi may utilize a VlsE-mediated system for immune avoidance of its surface antigens. To address this, the requirement of VlsE for host reinfection by the Lyme disease pathogen was investigated. Host-adapted wild type and VlsE mutant spirochetes were used to reinfect immunocompetent mice that had naturally cleared an infection with a VlsE-deficient clone. Our results demonstrate that variable VlsE is necessary for reinfection by B. burgdorferi, and this ability is directly related to evasion of the host antibody response. Moreover, the data presented here raise the possibility that VlsE prevents recognition of B. burgdorferi surface antigens from host antibodies. Overall, our findings represent a significant advance in our knowledge of immune evasion by B. burgdorferi, and provide insight to the possible mechanisms involved in VlsE-mediated immune avoidance.

Figure 1. Generation of B. burgdorferi mutant with non-switchable vlsE in cis.

A) Schematic illustrating the replacement of the vls locus with a non-switchable vlsE gene. The targeted deletion construct, pAR2, is inserted into lp28-1 via homologous recombination at the target DNA sequence. Following telomere resolution by endogenous ResT, the right end of lp28-1 containing the 15 silent cassettes and vlsE expression site is deleted. The resultant truncated plasmid, lp28-1Δvls::vlsE, contained vlsE with native basal promoter (denoted as p) and kanamycin-resistant gene (kan). The four leftward facing arrows represent the genes required for autonomous replication of lp28-1. Telomere regions and replicated telomere (rtel) are indicated as hatched regions. B) Analysis of the lp28-1Δvls::vlsE construct by field inversion gel electrophoresis. Genomic DNA from WT, ΔVlsE, and sVlsE are shown in lanes 1, 2, and 3, respectively. Positions of lp28-1Δvls::vlsE and lp28-1Δvls are shown by the arrowhead and angled line, respectively. The positions of DNA markers are indicated on the left.

Figure 2. Expression and surface localization of VlsE by B. burgdorferi clones.

Intact spirochetes were treated with or without proteinase K for 40 min followed by SDS-polyacrylamide gel electrophoresis (10⁸ cells/lane) and Western blotting. Two identical blots were processed with anti-VlsE or anti-FlaB antibodies. Lanes 1, 3, and 5 show that VlsE is only expressed by the WT and sVlsE clones, but not by the ΔVlsE clone. Treatment with proteinase K dramatically reduced VlsE immunostaining for the WT and sVlsE clones, but had no effect on levels of the periplasmic protein, FlaB. A non-specific band that remains after proteinase K digestion can also be seen in the anti-VlsE blot.

Figure 3. Experimental design to assay for a VlsE requirement for host reinfection.

C3H mice were initially infected with in vitro-grown ΔVlsE or sVlsE clones. At day 28 post infection, a time at which spirochetes had been cleared due to a host antibody response, animals were divided into three groups of 4 or 5 mice each and reinfected with either host-adapted WT, ΔVlsE, or sVlsE via tissue transplantation. Blood samples, ear biopsies and other harvested tissues (heart, bladder, and joint) were collected at indicated time points post transplantation and cultured to monitor the reinfection outcome.

Figure 4. Analysis of B. burgdorferi-specific immune sera by immunoblotting.

The whole-cell lysates of B. burgdorferi WT, sVlsE, and ΔVlsE clones (10⁶ cells/lane) were treated with anti-WT, anti-sVlsE, or anti-ΔVlsE (panel A, B, and C, respectively) immune sera collected from C3H mice at day 28 post infection. Preimmune sera-treated immunoblot had no immune banding (not shown).

Figure 5. Experimental design to assay for a VlsE requirement for evasion of B. burgdorferi-specific antibodies.

Immunologically-naïve SCID mice were injected with either WT-specific, ΔVlsE-specific, or preimmune sera. Mice were divided into groups of 3 animals each and challenged 18 hours later with host-adapted B. burgdorferi clones. Blood and other tissues were collected at day 7 post challenge and cultured for spirochetes. Group numbers are indicated in the parentheses.

Figure 6. Experimental design to assay for a VlsE requirement for evasion of T-cell independent antibodies.

Immunologically-naïve C3H mice were injected with either WT-specific, ΔVlsE-specific, or preimmune sera originated from nude mice. The sera-treated animals were divided into groups of 3 each and challenged 18 hours post-sera treatment with either host-adapted or in vitro-grown B. burgdorferi clones. Blood and other tissues were harvested at day 7 post challenge and cultured for spirochetes. Group numbers are indicated in the parentheses.