Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.

Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-β. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-β in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro-and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-β.Lyme disease | endosomal receptors | type I interferons | phagocytosis

Borrelia burgdorferi is the spirochetal agent of Lyme disease, a multisystemic disorder characterized by inflammation. Using global transcriptional profiling, we characterized the response of human PBMCs exposed to B. burgdorferi in an ex vivo coculture system. The expression profiles induced by B. burgdorferi were marked by the intense up-regulation of IFN-responsive transcripts and transcripts involved in the JAK/STAT signaling pathway. Transcript levels of IFN-α, IFN-β, and IRF7, and protein concentrations of IFN-α, were significantly elevated relative to those in unstimulated PBMCs. The induction of IFN-α was completely dependent upon phagocytosis of B. burgdorferi. Addition of a soluble type I IFN receptor, B18R, did not abolish the induction of IFN-inducible genes, indicating that B. burgdorferi directly elicits enhanced expression of these genes independently of type I IFN feedback signaling. Inhibitors of either TLR7 or TLR9 significantly reduced B. burgdorferi-stimulated IFN-α protein expression and transcription of IFN-induced genes. Simultaneous inhibition of both TLR7 and TLR9 completely abrogated IFN-α induction. The IFN-α-producing populations in PBMCs were identified as plasmacytoid dendritic and CD14+CD11c+ cells. These results reveal a TLR7/9-dependent signaling pathway used by human PBMCs to initiate a type I IFN response to the extracellular bacterium B. burgdorferi.

Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23°C and further enhanced by a temperature shift (37°C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by 70 and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by 70 exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod-and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.Lyme disease, the most common arthropod-borne infection in the United States, is caused by the spirochete Borrelia burgdorferi (87). In nature, B. burgdorferi has an obligate biannual enzootic cycle involving small mammalian host reservoirs, typically Peromyscus leucopus, and an Ixodes tick vector (51, 96). To successfully complete this life cycle, B. burgdorferi must adapt to and propagate within two markedly different physiologic milieus (67,75,96). A number of investigators have reported that manipulation of parameters, such as temperature, pH, DNA supercoiling, cell density, and partial O 2 pressure, during in vitro cultivation can trigger changes in gene and protein expression resembling those that occur when spirochetes adapt to their mammalian host (4,20,21,44,45,65,74,77,78,89,102). It als...

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Borrelia burgdorferi elicits a potent cytokine response through activation of multiple signaling receptors on innate immune cells. Spirochetal lipoproteins initiate expression of NF-B-dependent cytokines primarily via TLR2, whereas type I interferon (IFN) production is induced through the endosomal receptors TLR7 and TLR9 in human dendritic cells and TLR8 in monocytes. We demonstrate that DNA and RNA are the B. burgdorferi components that initiate a type I IFN response by human peripheral blood mononuclear cells (PBMCs). IFN-␣ protein and transcripts for IRF7, MX1, and OAS1 were induced by endosomal delivery of B. burgdorferi DNA, RNA, or whole-cell lysate, but not by lysate that had been treated with DNase and RNase. Induction of IFN-␣ and IFN-1, a type III IFN, by B. burgdorferi RNA or live spirochetes required TLR7-dependent signaling and correlated with significantly enhanced transcription and expression of IRF7 but not IRF3. Induction of type I and type III IFNs by B. burgdorferi RNA could be completely abrogated by a TLR7 inhibitor, IRS661. In addition to type I and type III IFNs, B. burgdorferi RNA contributed to the production of the NF-B-dependent cytokines, IFN-␥, interleukin-10 (IL-10), IL-1␤, IL-6, and tumor necrosis factor alpha (TNF-␣), by human PBMCs. Collectively, these data indicate that TLR7-dependent recognition of RNA is pivotal for IFN-␣ and IFN-1 production by human PBMCs, and that RNA-initiated signaling contributes to full potentiation of the cytokine response generated during B. burgdorferi infection.

The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi, the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ70-dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB, the glp operon, and bba74. As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB- and glp-gfp constructs containing only the minimal (−35/−10) σ70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between −165 and −82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential “gatekeeper” role of RpoS throughout the B. burgdorferi enzootic cycle.

This is the first study to globally assess the human skin transcriptional response during early Lyme disease. Borreliella burgdorferi elicits a predominant IFN signature in the EM lesion, suggesting a potential mechanism for spirochetal dissemination via IDO1-mediated localized immunosuppression.