Central role of the Holliday junction helicase RuvAB in vlsE recombination and infectivity of Borrelia burgdorferi

Abstract

Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa surface-exposed lipoprotein, undergoes antigenic variation during B. burgdorferi infection of mammalian hosts, and is believed to be a critical mechanism by which the spirochetes evade immune clearance. Random, segmental recombination between the expressed vlsE gene and adjacent vls silent cassettes generates a large number of different VlsE variants within the infected host. Although the occurrence and importance of vlsE sequence variation is well established, little is known about the biological mechanism of vlsE recombination. To identify factors important in antigenic variation and vlsE recombination, we screened transposon mutants of genes known to be involved in DNA recombination and repair for their effects on infectivity and vlsE recombination. Several mutants, including those in BB0023 (ruvA), BB0022 (ruvB), BB0797 (mutS), and BB0098 (mutS-II), showed reduced infectivity in immunocompetent C3H/HeN mice. Mutants in ruvA and ruvB exhibited greatly reduced rates of vlsE recombination in C3H/HeN mice, as determined by restriction fragment polymorphism (RFLP) screening and DNA sequence analysis. In severe combined immunodeficiency (C3H/scid) mice, the ruvA mutant retained full infectivity; however, all recovered clones retained the 'parental' vlsE sequence, consistent with low rates of vlsE recombination. These results suggest that the reduced infectivity of ruvA and ruvB mutants is the result of ineffective vlsE recombination and underscores the important role that vlsE recombination plays in immune evasion. Based on functional studies in other organisms, the RuvAB complex of B. burgdorferi may promote branch migration of Holliday junctions during vlsE recombination. Our findings are consistent with those in the accompanying article by Dresser et al., and together these studies provide the first examples of trans-acting factors involved in vlsE recombination.

Figure 1. RFLP analysis of vlsE cassette region PCR products indicates reduced vlsE variation in ruvA and ruvB mutants of B. burgdorferi.

Representative results obtained following inoculation of C3H/HeN mice with (A) the parental clone 5A18NP1, (B) the transposon mutant T11P01A01 (ruvA::himar1) and (C) T03TC051 (ruvB::himar1) are shown. Cultures from the indicated time points and tissues were used as the source of template DNA for amplification of the vlsE cassette region. The resulting PCR products were either treated (+) or not treated (-) with the restriction enzyme HphI. Presence of multiple bands or a smear in the HphI-treated sample is indicative of the presence of a high number of vlsE variants. The day post inoculation is indicated by D4 through D28. Tissues examined include skin (S), bladder (B), heart (H) and tibiotarsal joint (J).

Figure 2. vlsE sequence variation observed with the parental strain 5A18NP1 and the ruvA mutant T11P01A01 and the ruvB mutant T03TC051 following inoculation of immunocompetent C3H/HeN mice and immunodeficient C3H/scid mice.

The combined results from all tissues examined for each time point are shown. The vlsE cassette regions of individual B. burgdorferi clones were amplified by PCR, sequenced, and characterized as either identical to the parental vlsE sequence, a unique vlsE sequence for that mouse, or two or more clones with an identical vlsE variant sequence from a mouse (vlsE sequences with siblings).

Figure 3. Reduced vlsE sequence diversity generated following inoculation of C3H/HeN mice with the ruvA mutant T11P01A01.

Clones were isolated from C3H/HeN mice 28 days post inoculation with (A) the parental strain 5A18NP1 or (B) T11P01A01. The vlsE cassette region sequences of each clone were optimally aligned and then analyzed for sequence diversity using a phylogenetic tree program. The 5A18NP1 clones were isolated from a single mouse, whereas the T11P01A01 clones were obtained from all positive cultures of 6 inoculated mice. The groups of clones isolated from each mouse and their tissue source are indicated. In (A), brackets demarcate clusters of related (but often nonidentical) clones that occurred in the mouse infected with 5A18NP1. In the 5A18NP1 group, there were 31 unique vlsE sequences, 10 sequences with siblings, and 0 parental sequences. In (B), brackets indicate clones with identical vlsE sequences that were isolated from the 6 mice, frequently from multiple tissues. In this group of clones from mice inoculated with the ruvA mutant, there were 0 unique sequences, 8 sequences with many siblings, and 3 parental sequences. Trees are rooted with the 5A18NP1 parental vlsE sequence. A larger version of this figure is available at the website http://www.uth.tmc.edu/pathology/borrelia/.

Figure 4. Low complexity of vlsE recombination events in ruvA and ruvB mutants.

The positions of the six variable regions (VR1–VR6) and six relatively invariant regions (IR1–IR6) within the vlsE central cassette region are provided at the top of the figure. The locations and silent cassette sources of the most likely recombination events for each clone depicted are indicated. A representative clone from a 28 day infection with the parental strain 5A18NP1 is shown at the top. The remainder of the figure shows the vlsE variant clonotypes isolated from C3H/HeN mice 28 days post inoculation with the ruvA mutant T11P01A01 or the ruvB mutant T03TC051. The possible involvement of each of the silent cassettes in sequence variation was analyzed using an Excel®/Visual Basic program, as described previously and depicted in Fig. S2. The horizontal colored bars represent regions of each silent cassette (vls2 to vls16, top to bottom) that match the sequence changes found in the variant clone. Dark regions in each bar correspond to the actual sequence changes, whereas the lighter portion of the bar represents the maximum possible region of that silent cassette exchanged into vlsE to produce the observed sequence change. In isolates from Animal 2 inoculated with the ruvA mutant, a 9 bp untemplated change (black hatched box) that did not match the vls silent cassettes or any other genomic sequence was present in all clones isolated.

Figure 5. The ruvA mutant T11P01A01 does not exhibit enhanced sensitivity to UV light or mitomycin C.

(A) Survival of ruvA mutant and parental strain 5A18NP1 after exposure of UV light. The dosages of UV irradiation (254 nm) applied to the bacterial suspensions are indicated. (B) Survival of ruvA mutant and parental strain 5A18NP1 after 14 h treatment with the indicated doses of mitomycin C in BSK II medium. Colony counts on duplicate or triplicate plates were utilized to determine the concentration of surviving B. burgdorferi. Results shown are for representative experiments from three independent studies for each treatment.