The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host's acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA's dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5' end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions.

SummaryThe Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multisystemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.

Edited by Chris WhitfieldLyme disease, also known as Lyme borreliosis, is the most common tick-transmitted disease in the Northern Hemisphere. The disease is caused by the bacterial spirochete Borrelia burgdorferi and other related Borrelia species. One of the many fascinating features of this unique pathogen is an elaborate system for antigenic variation, whereby the sequence of the surfacebound lipoprotein VlsE is continually modified through segmental gene conversion events. This perpetual changing of the guard allows the pathogen to remain one step ahead of the acquired immune response, enabling persistent infection. Accordingly, the vls locus is the most evolutionarily diverse genetic element in Lyme disease-causing borreliae. Small stretches of information are transferred from a series of silent cassettes in the vls locus to generate an expressed mosaic vlsE gene version that contains genetic information from several different silent cassettes, resulting in ϳ10 40 possible vlsE sequences. Yet, despite its extreme evolutionary flexibility, the locus has rigidly conserved structural features. These include a telomeric location of the vlsE gene, an inverse orientation of vlsE and the silent cassettes, the presence of nearly perfect inverted repeats of ϳ100 bp near the 5 end of vlsE, and an exceedingly high concentration of G runs in vlsE and the silent cassettes. We discuss the possible roles of these evolutionarily conserved features, highlight recent findings from several studies that have used next-generation DNA sequencing to unravel the switching process, and review advances in the development of a mini-vls system for genetic manipulation of the locus.

In Azotobacter vinelandii the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic algD mRNA, acting as a translational repressor, and GacA was found to activate transcription of the rsmZ1 and rsmZ2 genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one rsmY and seven rsmZ1-rsmZ7 genes present in the A. vinelandii genome, and that inactivation of rsmA results in increased PHB production. Transcriptional and translational phbR-gusA gene fusions were used to show that the gacA mutation negatively affected the expression of the phbR gene at the translational level. We also demonstrated an in vitro interaction of RsmA with RNAs corresponding to phbB and phbR mRNA leaders, and showed that the stability of phbR and phbB mRNAs is increased in the rsmA mutant. Taken together these results indicate that in A. vinelandii, RsmA post-transcriptionally represses the expression of PhbR.