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.

Figure 1. Gene disruption and confirmation.

A) Gene disruption strategy. The infectious B. burgdorferi strain B31, clone 5A4 (B31-5A4) was transformed with a knockout plasmid carrying a one kb gentamicin cassette (blue) that replaced the central portion of the target gene (yellow) as described in Materials and Methods. The two possible outcomes of recombination events with the target gene are shown: allelic exchange would result in gene disruption while integrative recombination of the knockout plasmid would result in merodiploid formation. The position of PCR primers used for construct verification are shown by arrows on the schematic. B) Construct verification of the mutL disruption by PCR. Each gene disruption was subjected to four PCR analyses. 1) The presence of the gentamicin resistance cassette was confirmed as shown in lanes 1 and 2. The shuttle vector pBSV2G served as the positive control c⁺ for amplification of the gent cassette (lane 3.) 2) The portion of mutL expected to be deleted in a gene disruption was not detected in either mutL1 or 2 (lanes 5 and 6); however, it was detected in the positive control (c+), which contained wild-type B31-5A4 DNA as a template in lane 7. 3) The size of the target gene was compared in mutL1 and 2 genotypes. The expected 2.1 kb products for a gene disruption were observed (lanes 9 and 10) in comparison to the 1.5 kb product from the mutL⁺ genotype (lane 11). Lanes 4, 8 and 12 are negative controls (c⁻) that lacked DNA template. 4) Confirmation of the correct insertion site was performed using combinations of the target gene primers and primers internal to the gentamicin cassette to amplify the boundaries. The left boundary in both mutL clones gave the expected 0.55 kb product (lanes 13 and 15). The right boundary in both clones gave the expected product of approximately 1.3 kb (lanes 14 and 16). A 100bp ladder on the left side, relevant to the two left panels, and a 1kb ladder on the right side, which applies to the two right panels, were the molecular weight markers (M) used.

Figure 2. Restriction fragment length polymorphism assay for switching at vlsE.

A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2/1 DNA templates are found in lanes 5 & 6, and 7 & 8 respectively. M denotes a 100bp molecular weight marker.

Figure 3. Number of switched vlsE clones in SCID C3H/HeN mice.

Sequencing of the cloned PCR product of the vlsE variable regions using primer pJET1.2/forward was performed on 10 clones from each tissue type culture for each genotype (see Materials and Methods). The y-axis denotes the number of clones out of ten that contained templated nucleotide changes in variable regions 1–6 (switches) and the x-axis denotes the tissue type. The P-values above the bars indicate the level of significance of the difference between the wild-type and mutant samples, calculated using Fisher's Exact test.