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.

Significance Invariant natural killer T cells (iNKT) have been found primarily patrolling inside blood vessels in the liver, where they respond to bacterial glycolipids presented by CD1d on liver macrophages. We show joint iNKT cells are localized outside of blood vessels and respond directly to the joint-homing pathogen, Borrelia burgdorferi , which causes Lyme borreliosis using multichannel spinning-disk intravital microscopy. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted its dissemination attempts into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway. These results suggest a critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier.

Spirochetes causing Lyme borreliosis are obligate parasites that can only be found in a tick vector or a vertebrate host. The ability to survive in these two disparate environments requires up and downregulation of specific genes by regulatory circuits that remain largely obscure. In this work on the Lyme spirochete, B. burgdorferi, we show that a disruption of the hrpA gene, which encodes a putative RNA helicase, results in a complete loss in the ability of the spirochetes to infect mice by needle inoculation. Studies of protein expression in culture by 2D gels revealed a change in the expression of 33 proteins in hrpA clones relative to the wild-type parent. Quantitative characterization of protein expression by iTRAQ analysis revealed a total of 187 differentially regulated proteins in an hrpA background: 90 downregulated and 97 upregulated. Forty-two of the 90 downregulated and 65 of the 97 upregulated proteins are not regulated under any conditions by the previously reported regulators in B. burgdorferi (bosR, rrp2, rpoN, rpoS or rrp1). Downregulated and upregulated proteins also fell into distinct functional categories. We conclude that HrpA is part of a new and distinct global regulatory pathway in B. burgdorferi gene expression. Because an HrpA orthologue is present in many bacteria, its participation in global regulation in B. burgdorferi may have relevance in other bacterial species where its function remains obscure. We believe this to be the first report of a role for an RNA helicase in a global regulatory pathway in bacteria. This finding is particularly timely with the recent growth of the field of RNA regulation of gene expression and the ability of RNA helicases to modulate RNA structure and function.

To survive and avoid accumulation of mutations caused by DNA damage, the genomes of prokaryotes encode a variety of DNA repair pathways most well characterized in Escherichia coli. Some of these are required for the infectivity of various pathogens. In this study, the importance of 25 DNA repair/recombination genes for Borrelia burgdorferi survival to UV-induced DNA damage was assessed. In contrast to E. coli, where 15 of these genes have an effect on survival of UV irradiation, disruption of recombinational repair, transcription-coupled repair, methyl-directed mismatch correction, and repair of arrested replication fork pathways did not decrease survival of B. burgdorferi exposed to UV light. However, the disruption of the B. burgdorferi nucleotide excision repair (NER) pathway (uvrA, uvrB, uvrC, and uvrD) resulted in a 10-to 1,000-fold increase in sensitivity to UV light. A functional NER pathway was also shown to be required for B. burgdorferi resistance to nitrosative damage. Finally, disruption of uvrA, uvrC, and uvrD had only a minor effect upon murine infection by increasing the time required for dissemination.

The Lyme disease spirochete Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA (bb0603 [p66], bba74, bb0241 [glpK], bb0242 and bb0243 [glpA]) suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.