Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch Migrase

Abstract

Persistent infection by pathogenic organisms requires effective strategies for the defense of these organisms against the host immune response. A common strategy employed by many pathogens to escape immune recognition and clearance is to continually vary surface epitopes through recombinational shuffling of genetic information. Borrelia burgdorferi, a causative agent of Lyme borreliosis, encodes a surface-bound lipoprotein, VlsE. This protein is encoded by the vlsE locus carried at the right end of the linear plasmid lp28-1. Adjacent to the expression locus are 15 silent cassettes carrying information that is moved into the vlsE locus through segmental gene conversion events. The protein players and molecular mechanism of recombinational switching at vlsE have not been characterized. In this study, we analyzed the effect of the independent disruption of 17 genes that encode factors involved in DNA recombination, repair or replication on recombinational switching at the vlsE locus during murine infection. In Neisseria gonorrhoeae, 10 such genes have been implicated in recombinational switching at the pilE locus. Eight of these genes, including recA, are either absent from B. burgdorferi, or do not show an obvious requirement for switching at vlsE. The only genes that are required in both organisms are ruvA and ruvB, which encode subunits of a Holliday junction branch migrase. Disruption of these genes results in a dramatic decrease in vlsE recombination with a phenotype similar to that observed for lp28-1 or vls-minus spirochetes: productive infection at week 1 with clearance by day 21. In SCID mice, the persistence defect observed with ruvA and ruvB mutants was fully rescued as previously observed for vlsE-deficient B. burgdorferi. We report the requirement of the RuvAB branch migrase in recombinational switching at vlsE, the first essential factor to be identified in this process. These findings are supported by the independent work of Lin et al. in the accompanying article, who also found a requirement for the RuvAB branch migrase. Our results also indicate that the mechanism of switching at vlsE in B. burgdorferi is distinct from switching at pilE in N. gonorrhoeae, which is the only other organism analyzed genetically in detail. Finally, our findings suggest a unique mechanism for switching at vlsE and a role for currently unidentified B. burgdorferi proteins in this process.

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

“…The activity of RuvAB has also been implicated in the antigenic variation systems of vlsE in Borrelia burgdorferi and pilE in Neisseria gonorrhoeae, both of which depend upon recombination (44)(45)(46)(47). In the case of N. gonorrhoeae, another branch migration helicase, RecG, has been implicated (47).…”

Construction and Characterization of a Borrelia burgdorferi Strain with Conditional Expression of the Essential Telomere Resolvase, ResT

“…B. burgdorferi was grown in BSK II medium (44) prepared in-house and supplemented with 5% rabbit serum (Cedar Lane). B. burgdorferi transformations were performed as described previously (45,46). PCR was performed on genomic DNA to screen for the presence of the insert.…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…To date, however, the machinery needed for an HR reaction of such a short length is unknown. Gene conversion during antigenic variation that is independent of the HR recombinase is not unique to T. brucei: Borrelia burgdorferi is a tick-borne spirochete where recombination of silent vls gene segments into the single vlsE expression site is unaffected by mutation of RecA (169), though it does require RuvAB, a helicase that processes Holliday junction HR intermediates (170,171). Like T. brucei, how homology-directed strand transfer initiates in B. burgdorferi without RecA/RAD51 is unknown, though the potential involvement of a novel, telomere-directed resolvase would be very interesting (172).…”

“…Elevated switching by crossover in T. brucei RTR mutants (117,145) provides evidence not only that reciprocal recombination can occur but also that it is normally suppressed between BES. However, the formation and resolution of Holliday junction intermediates has not been demonstrated, such as by testing for a role of Holliday junction resolvases (219), which have been shown to act in both N. gonorrhoeae and B. burgdorferi antigenic variation (131,170,171). It should be noted that it remains unclear whether any of the above reactions can account for VSG SGC, and this will be an important next phase of investigation, given the importance of this reaction to the parasite.…”