Toll-Like Receptors 1 and 2 Heterodimers Alter Borreliaburgdorferi Gene Expression in Mice and Ticks
Erol Fikrig1,2, Sukanya Narasimhan1, Girish Neelakanta1, Utpal Pal1,a, Manchuan Chen3,and Richard Flavell2,31Section of Infectious Diseases, Department of Internal Medicine, New Haven, Connecticut2Howard Hughes Medical Institute, New Haven, Connecticut3Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut
AbstractBorrelia burgdorferi, the agent of Lyme disease, is recognized by Toll-like receptor (TLR) 1 and 2heterodimers. Microarray analysis of in vivo B. burgdorferi gene expression in murine skin showedthat several genes were altered in TLR1/2-deficient animals compared with wild-type mice. Forexample, expression of bbe21 (a gene involved in B. burgdorferi lp25 plasmid maintenance) andbb0665 (a gene encoding a glycosyl transferase) were higher in TLR1/2-deficient mice than in controlanimals. In contrast, messenger RNA levels for bb0731 (a spoJ-like gene) and bba74 (a geneencoding a periplasmic protein) were lower in TLR1/2-deficient mice than in wild-type animals. Theexpression profiles of some of these genes were altered similarly in B. burgdorferi– infected ticksfed on control or TLR1/2-deficient mice. Quantitative reverse-transcription polymerase chainreaction analysis supported the microarray analysis and suggested that spirochete gene expression isaltered by the milieu created by specific host TLRs, both in the murine host and in the arthropodvector.
Lyme disease, which is caused by Borrelia burgdorferi, is the most common tickborneinfectious disease in the United States [1,2]. Pathognomonic rash, arthritis, and carditis arecommon clinical manifestations [1,2]. Murine models partially mimic the human illness, giventhat the animals develop a persistent infection and tissue inflammation, and host immuneresponses are critical for controlling disease [3].
The surface of B. burgdorferi is unique and does not befit its apparently gram-negative cellwall [4]. The segmented genome of B. burgdorferi encodes ~130–150 lipoproteins [5,6], manyof which decorate the outer wall of the spirochete. Surface proteins of the spirochete presenta critical interface between the bacterium and its diverse niches in the vertebrate andinvertebrate host. The spirochete apparently changes its transcriptome to successfullydisseminate and survive in the different niches [7]. B. burgdorferi surface proteins possiblylead these changes via direct interactions with the microenvironment, as seen by signaturechanges in the makeup of the outer surface of the spirochete that precede and succeed itsmigration to specific niches [7]. However, in their vantage position, the outer surface proteinsof Borrelia also risk hostile interactions with host innate immune molecules.
NIH Public AccessAuthor ManuscriptJ Infect Dis. Author manuscript; available in PMC 2010 March 27.
Published in final edited form as:J Infect Dis. 2009 October 15; 200(8): 1331–1340. doi:10.1086/605950.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Toll-like receptors (TLRs) interact with a wide variety of microbial molecules and are criticalfor initiating host defenses [8–10]. The triacylated lipoproteins on the surface of the Lymedisease agent are recognized by TLR1/2 heterodimers, resulting in the activation of innateimmune signaling pathways [11,12]. This reduces spirochete infection in mice, because higherpathogen loads are evident in mice lacking either TLR1 or TLR2 when challenged with variousB. burgdorferi isolates [11,13–16]. Furthermore, myeloid differentiation primary responseprotein 88 (MyD88)–deficient mice have greater difficulty controlling spirochetes than doTLR1- or TLR2-deficient animals [17–19]. MyD88 serves as the adapter molecule for severalTLRs, suggesting that additional receptors are also involved in the identification of B.burgdorferi.
The spirochete uses several strategies to circumvent these immune responses. When it entersthe host via a tick bite, B. burgdorferi begins its standoff with the host immune responses byexploiting tick salivary proteins spit into the bite site [20,21]. Soon after entry into the host,the spirochete transcriptome and proteome undergo further changes as they adapt in the host,using their own proteins (such as the complement regulator– acquiring surface proteins) todefuse [22] and extra-cellular matrix binding-proteins to escape [23] host immune reactions.Liang et al [24] have suggested that the expression profile of several Borrelia genes—includingospc, bbf01, and vlsE [25]—might be altered by host humoral responses. Crowley and Hubner[26] have shown that an in vivo inflammatory environment induces the expression of outersurface protein A by unknown molecular mechanisms. Work by Anguita et al [27] has indicatedthat the recombination of the vlsE locus, an important aspect of immune evasion, might beinfluenced by interferon γ–mediated inflammation in the host. Because the engagement ofspirochete lipoproteins with TLRs is a critical initiator of host immune responses to Borrelia[28], TLR-mediated signals might influence spirochete gene expression. In the present study,we examined how gene expression in the Lyme disease agent is altered by TLR1/2-mediatedsignals, using TLR1/2 heterodimer recognition of B. burgdorferi as a model.
METHODSB. burgdorferi, mice, and Ixodes scapularis
Virulent, low-passage, clonal B. burgdorferi N40 was used. TLR1/2 heterodimers cannot formin TLR1/2-, TLR2-, or TLR1-deficient animals. The functional defect in these animals issimilar because TLR1/2 heterodimers are required for the recognition of B. burgdorferilipoproteins. TLR1/2- and TLR1-deficient mice on a B6 background were used in allexperiments [11], and we collectively refer to the TLR1/2 heterodimer–deficient animals asTLR1/2-deficient mice. Control B6 mice were purchased from the National Institutes of Health.Mice were infected by means of a single subcutaneous inoculum of 1 × 104 spirochetes; 2weeks later, mice were killed and tissue specimens collected. For inoculation of mice withhost-adapted spirochetes to assess the virulence of B. burgdorferi, donor B6 wild-type andTLR1/2-deficient mice (5 animals in each group) were infected by means of needle inoculation,as described above. Two weeks after infection, ear punch samples from TLR1/2- deficient andwild-type mice were implanted into the skin of naive wild-type B6 mice (5 mice in each group),as described elsewhere [24]. Mice were killed 14 days after infection, and bladders and skinwere collected for DNA preparation and for quantitative polymerase chain reaction (PCR).
B. burgdorferi–infected I. scapularis nymphs were maintained at a tick colony at Yale. Eightto 10 B. burgdorferi N40–infected I. scapularis nymphs were placed on each control orTLR1/2-deficient mouse (5 mice in each group), and ticks were fed to repletion. Guts andsalivary glands dissected from fed ticks were pooled into groups of 5 ticks for RNA extractionand complementary DNA (cDNA) synthesis, as described elsewhere [29]. Guts from unfedN40-infected nymphs were also processed for cDNA synthesis, as described above.
Fikrig et al. Page 2
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Microarray analysisAmplification of spirochete transcripts in infected mice was performed using DECAL(differential expression analysis using a custom-amplified library), as described elsewhere[30]. Groups of 5 TLR1/2-deficient and control mice were injected with 1 × 104 B.burgdorferi per mouse, and infection was confirmed by PCR or culture. Animals were killedon day 14 after spirochete challenge. Positive selection, amplification of spirochete transcripts,normalization, and random-prime labeling to generate biotin-labeled probes were done asdescribed elsewhere [30]. The normalized probes were used to hybridize duplicate B.burgdorferi whole-genome nylon membrane arrays [31]. After the arrays were probed withDECAL-enriched cDNA, the hybridization was scored visually by 3 independent observers,and spots corresponding to gene elements were given a score from 0 to 3 (in increments of0.5), on the basis of the intensity of hybridization [30]. A gene was considered differentiallyexpressed when at least a 2-fold increase or decrease in hybridization score was noted (eg,from 1.5 to 3.0 or from 1 to 2) between experimental and control arrays. The expression offlaB was unchanged, as judged by a score of 0.5 in both the arrays. Spot pairs correspondingto differentially expressed genes were quantitatively analyzed using ImageJ software (version1.32j), a public-domain image-processing program (available at: http://rsb.info.nih.gov/ij). Thearrays were scanned using an HP LaserJet4010 scanner and were saved as an uncompressedTIFF image. Pixel values for the selected spot pairs were then measured in ImageJ. Ratios ofthe mean pixel intensities of experimental spot pairs and respective control spot pairs werenormalized to the pixel intensity ratios of flaB in each array, to derive normalized fold-changevalues for gene expression. Digital analysis was congruent with the visual analysis.
PCRB. burgdorferi–infected murine tissues and spirochete-infected ticks were dissected, and totalRNA was processed for quantitative reverse-transcription PCR using the iQ SYBR GreenSupermix (Bio-Rad), as described elsewhere [29]. The following primers were used: for tickactin, 5′-GGCGACGTAGCAG-3′ (forward) and 5′-GGTATCGTGCTCGACTC-3′ (reverse);for flaB, 5′-TTCAATCAGGTAACGGCACA-3′ (forward) and 5′-GACGCTTGAGACCCTGAAAG-3′ (reverse); for bbe21, 5′-AAACCATCCGAAGTTGAGGAGG-3′ (forward) and 5′-ACTTCTTTTTGCCCGTTGCG-3′ (reverse); for mouse β-actin, 5′-TCACCCACACTGTGCCCATCTACGA-3′ (forward) and 5′-GGATGCCACAGGATTCCATACCCA-3′ (reverse); for bb0665, 5′-ACAGCTTGGAGGTTTTGACG-3′ (forward) and 5′-AAAACAACGCCATTTCCAAC-3′(reverse); for bba74, 5′-CTCAAGCCTCAATCCAATGTTTTAGA-3′ (forward) and 5′-CTACTCCTGCATCATTTGATTCTACCA-3′ (reverse); and for bb0731, 5′-GGAGACATTGAAACTCTTAAAAACA-3′ (forward) and 5′-TTGCCTCGCTTCTTGTGAAT-3′ (reverse). The amount of B. burgdorferi transcripts in eachsample was normalized to levels of flaB transcripts. The murine or tick β-actin gene, dependingon the sample, was amplified to normalize the amount of B. burgdorferi cDNA [29,32]. Datawere analyzed using Excel (version 11.5.2; Microsoft) or Prism (version 4; Graphpad Software)software, and results were expressed as means ± standard errors. The significance of thedifferences between mean values was assessed by 2-tailed Student t test.
Plasmid profile analysisBoth wild-type and TLR1/2-deficient mice were injected by needle with 1 × 104 in vitro–cultivated spirochetes, as described above. Fourteen days after infection, bladders wereharvested and processed for DNA extraction using a DNeasy kit (Qiagen). Plasmid profileswere analyzed using the primers described by Parveen et al [33], and levels were normalizedto those of flaB.
Fikrig et al. Page 3
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
In silico analysisDNA and protein sequences of B. burgdorferi genes were obtained from the ComprehensiveMicrobial Resource Web site (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi).Homology searches and protein subcellular location predictions were conducted using theBLAST (http://blast.ncbi.nlm.nih.gov) and PSORTb (http://www.psort.org/psortb) analysistools, respectively.
RESULTSAlteration of spirochete gene expression in B. burgdorferi–infected TLR1/2-deficient mice
TLR1 and TLR2 recognize B. burgdorferi lipoproteins, thereby initiating a signaling cascadethat results in host cell activation and cytokine release. We determined here whether TLR-mediated responses, as a counterbalance, influence pathogen gene expression. B.burgdorferi microarrays were used to examine spirochete mRNAs in the skin during infectionof TLR1/2-deficient and control mice.
The B. burgdorferi gene expression profile 2 weeks after infection in TLR1/2-deficient andcontrol mice was mostly similar, at least within the limitations of the capabilities of themicroarray and DECAL procedures. However, a small subset of genes were differentiallyexpressed (Figure 1). Approximately 20 spirochete genes demonstrated enhanced expression(on the basis of an increase of 1.5-fold or more in hybridization score, as judged by visual anddigital analysis of pixel intensities) in TLR1/2-deficient animals compared with wild-type mice(Table 1). Genes that showed increased expression in wild-type mice (increase of 1.5-fold ormore in hybridization score) compared with TLR1/2-deficient animals were also apparent(Table 2). A second separate experiment yielded the same result. Two up-regulated and 2 down-regulated genes in TLR1/2-deficient mice were selected to establish the paradigm that TLR1/2-mediated responses alter spirochete gene expression. To examine the effect that the hostimmune status has on both tick and vertebrate host milieus, we prioritized genes on the basisof our ability to unambiguously detect their expression in N40-infected murine and tick host.Genes that belonged to large paralogous families were not prioritized, because sequenceidentity often confounds PCR and array analysis. On the basis of these criteria, we focused onbbe21 and bb0665, which showed markedly increased expression in TLR1/2-deficient animalscompared with wild-type mice during murine Lyme borreliosis, and on bb0731 and bba74,which showed markedly decreased expression.
Increased expression of B. burgdorferi bbe21 and bb0665 and decreased expression ofbb0731 and bba74 in TLR1/2-deficient mice
In each experiment, 5 TLR1/2-deficient mice and 5 control mice were challenged with B.burgdorferi, and each mouse was individually examined for spirochete gene expression.Consistent with earlier observations [13], the B. burgdorferi burden was higher in TLR1/2-deficient mice than in control mice (Figure 2A), and joint inflammation was evident in all theanimals. At 2 weeks, when spirochete numbers are high in the dermis, B. burgdorferi bbe21and bb0665 mRNA levels were significantly higher in the skin of TLR1/2-deficient mice thanin wild-type mice (P < .05) (Figure 2B). In contrast, bb0731 and bba74 levels were significantlydecreased in TLR1/2-deficient animals compared with control mice (P < .05) (Figure 2B). Thisexpression profile was also observed in the bladder (Figure 2C), and differences in theexpression of all 4 genes were statistically significant. These results confirmed the initialmicroarray results (Table 1 and Table 2).
Fikrig et al. Page 4
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
No alteration in plasmid profile and infectivity of spirochetes in TLR1/2-deficient miceAltered B. burgdorferi gene expression could reflect a population of avirulent spirochetes thathad been enriched in the TLR1/2-deficient host because of dampened immune responses. Toaddress this, we first examined the plasmid profile of B. burgdorferi cultured from the bladdersof wild-type and TLR1/2-deficient animals. Spirochetes from both control and experimentalanimals were positive by PCR for all the detectable plasmids assessed (data not shown). Levelsof lp25, a plasmid critical for spirochete virulence in both mammals and ticks [7], were similarin control and experimental groups and were comparable with the virulent low-passage N40strain used in this study (Figure 3A). Furthermore, we grafted skin from B. burgdorferi–infectedTLR1/2-deficient and wild-type mice onto naive wild-type mice. We reasoned that if theTLR1/2-deficient mice were enriched for avirulent spirochetes, then these B. burgdorferiwould be eliminated in the wild-type host. Mice that received skin grafts from TLR1/2-deficientmice demonstrated spirochete burdens comparable to those in mice that received skin graftsfrom wild-type mice (Figure 3B).
Lower B. burgdorferi bba74 and bb0731 expression in ticks fed on TLR1/2-deficient miceWe then determined whether TLR1 and TLR2 influenced bbe21, bb0665, bb0731, andbba74 mRNA levels in the vector by allowing ticks to engorge on TLR1/2-deficient or wild-type mice. The B. burgdorferi burden in the midguts of ticks fed on TLR1/2-deficient animalswas significantly higher than the spirochete burden in ticks fed on control animals (Figure4A). Spirochetes made substantially less bbe21, bb0731, and bba74 mRNA, but not bb0665mRNA, in unfed ticks than in engorged vectors (Figure 4B–4E). B. burgdorferi bbe21 andbb0665 expression in the midguts of ticks was not altered by TLR1/2 deficiency of the murinehost (Figure 4B and 4C). Expression of bba74 and bb0731 was significantly lower in themidguts of ticks fed on TLR1/2-deficient mice than in ticks fed on control animals (Figure4D and 4E). Expression of bbe21 and bb0665 was increased and expression of bba74 andbb0731 was decreased in the salivary glands of ticks fed on TLR1/2-deficient mice comparedwith that in ticks fed on control animals. The expression levels of all 4 genes examined in thisstudy were ~500–1000-fold higher in nymphal ticks compared with those in the murine host.
DISCUSSIONSpirochete-mediated stimulation of TLR1 and TLR2 causes a cascade of events that results inthe generation of cellular and humoral immune responses that diminish B. burgdorferi burdenin vivo [13]. The roles played by TLR2, TLR5, TLR9, and the major TLR adaptor MyD88 inthe clearance of Borrelia has been defined [34]. While earlier efforts suggested that Borreliagene expression may be modulated by humoral immunity [35], studies have not determinedwhether pathogen gene expression in vivo is altered by key components of host innateimmunity, such as TLRs. We observed that the expression of a subset of spirochete genes wereenhanced or decreased in TLR1/2-deficient animals compared with wild-type mice (Table 1and Table 2). TLR1/2 deficiency did not predominantly influence the expression of Borreliagenes encoding lipoproteins, the predominant ligands for TLR1/2 activation [28]. Differencesin the humoral responses apparently modified the expression of several lipoprotein-encodinggenes, including ospC, bbf01, and vlsE [24,25]. The present study used a TLR1/2-deficienthost, but Borrelia components also engage with other TLRs [36] and would continue to triggerthe other TLRs, as in the wild-type host. We chose to examine bbe21, bb0665, bb0731, andbba74 expression profiles in greater detail, because mRNA levels of these genes wereconsistently altered in replicate experiments in TLR1/2-deficient mice compared with controlsand because their expression is readily detectable in the tick vector.
The gene bbe21, a member of the paralogous family 57, may participate in plasmid replication[6,37]. Stewart et al [38] have also suggested a role for BBE21 in the replication of lp25. lp25
Fikrig et al. Page 5
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
is essential for Borrelia survival in both the vector and host milieus [7], providingnicotinamidase (encoded by the gene pncA [BBE22]), a key enzyme for the synthesis of thecofactor nicotinamide adenine dinucleotide [39]. We observed bbe21 up-regulation coincidentwith active spirochete replication in the arthropod and the TLR1/2-deficient mammalian host.Whether the preferential increase in bbe21 expression in TLR1/2-deficient mice relates toincreased spirochete burden remains to be determined.
The gene bb0665, a chromosomal gene, encodes for a 32-kDa protein predicted to contain aputative glycosyl transferase domain that might be involved in the transfer of sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range ofsubstrates (http://cmr.jcvi.org/tigr-scripts/CMR/CmrHomePage.cgi). Borrelia also containsseveral low-molecular-weight glycosylated lipids that are potentially immunogenic [28,40,41]. The decrease in bb0665 expression in control mice compared with TLR1/2-deficient mice(Figure 2) might help the spirochetes to remain less “visible” to the host immune surveillance.More recently, Yang and Li [42] have shown that bb0665, along with bb0666 and bb0667, ispart of an operon termed pami. It has been suggested that bb0666 encodes a putative N-acetylmuramyl-L-alanine amidase involved in peptidogylcan synthesis critical for cell division[42]. The product of bb0665, as part of the pami operon, could be involved in transferringsugars to the peptidoglycan layer during cell wall biosynthesis and cell division. One wouldexpectedly observe increased bb0665 expression in TLR1/2-deficient mice, in which thespirochete burden is increased compared with control mice.
The gene bba74, which is contained on the linear plasmid 54, encodes for a 257-aa proteinrecently shown to be a periplasmic protein associated with the outer membrane [43]. Althoughbba74 expression was increased in vitro when Borrelia was temperature shifted from 23°C to37°C [31,44], it was repressed under mammal-like conditions simulated using the dialysismembrane chamber implant model [45] and in vitro by increasing the temperature in thepresence of blood in the culture medium [46]. Mulay et al [47] have demonstrated thatbba74 expression, like that of ospA, is turned off in the murine host, repression that is apparentlymediated by the Borrelia alternative sigma factor RpoS. In the present study, we observedbba74 transcripts in the murine host, although the expression levels of bba74 decreased ~1000-fold in the murine host compared with that in fed nymphs (Figure 2). Although the presentstudy used B6 mice, Mulay et al [47] used C3H/HeJ mice. Unlike B6 mice, C3H/HeJ miceencode a defective TLR4 protein, which leads to an impaired TLR4 response [48] that couldaccount for the observed difference between the 2 studies. Interestingly, expression of bba74was reduced ~50-fold in TLR1/2-deficient B6 mice compared with that in wild-type B6 mice(Figure 2), suggesting that bba74 expression may be additionally influenced by the immunestatus of the host.
The gene bb0731, a chromosomal gene, encodes for a predicted 13-kDa protein that containsdomains related to the plasmid partitioning the protein ParB and that is a paralogue of theBorrelia gene bb0434, which encodes the stage 0 sporulation protein J (Spo0J). The Spo0Jfamily of proteins are thought to play a vital role in coupling developmental transcription tocell cycle progression, acting as a biological switch regulating vegetative growth andsporulation [49]. Borrelia does not sporulate; however, its transcriptional status changes indifferent milieus, remaining quiescent (as in unfed tick guts) or actively replicating (as infeeding ticks) [50]. The increased levels of bb0731 transcripts in immunocompetent controlmice compared with TLR1/2-deficient mice might be inversely coupled to the spirochete’sability to replicate actively in response to altered environment.
Grafting of skin from TLR1/2-deficient mice onto wild-type mice produced spirochete burdenscomparable to those observed in mice that received skin grafts from wild-type mice (Figure3B). This finding demonstrated that spirochetes in TLR1/2-deficient mice were virulent and
Fikrig et al. Page 6
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
ruled out the possibility that differential expression of spirochete genes in TLR1/2-deficientanimals was not an artifact of selection of spirochetes expressing a set of preferred genes.
Spirochetes in the vector are passively exposed to the TLR receptors and to host immunemolecules (including cytokines) when ticks take a blood meal. We therefore examined whetherthe TLR1/2-deficient blood meal would influence the expression of bbe21, bb0665, bba74,and bb0731 within the tick. Expression of bbe21 and bb0665 in the tick gut did not changesignificantly when ticks were fed on TLR1/2-deficient or control animals (Figure 4B and4C). This departure from observations made in the murine host suggests that the role playedby arthropod-specific signals in the expression of these genes in the tick gut is predominant.However, the decrease in bba74 and bb0731 transcripts in nymphs fed on TLR1/2-deficientmice compared with nymphs fed on wild-type mice indicates the potential influence that thehost immune system has on the expression of bba74 in the tick gut (Figure 4D and 4E).Expression levels of bbe21 and bb0665 were increased and expression levels of bba74 andbb0731 were decreased in the salivary glands of ticks fed on TLR1/2-deficient mice (Figure4B–4E), a trend that mirrors the profiles observed in the murine host (Figure 2). Theseobservations reveal a facet of host-pathogen interactions that occurs within the vector, perhapsto better prepare the outgoing pathogen for optimal survival in the competent host.
The interplay between pathogens and their vertebrate and invertebrate hosts is complex. Thepresent study shows that B. burgdorferi gene expression is influenced by specific TLR-mediated innate immune responses, perhaps to enhance spirochete survival. Although a decadehas passed since the sequencing of the genome of B. burgdorferi [6], the functions of the majorportion of the genome are not understood. This limits our ability to infer the physiologicalsignificance of differential expression of genes and frustrates transcriptome analysis ofBorrelia. Despite this limitation, a theme appears to emerge from the in silico analysis of the4 genes differentially expressed in TLR1/2-deficient mice. The products of the genesbb0665, bb0731, and bbe21, but not bba74, appear to be involved in the modulation ofspirochete replication, consistent with the obvious differences in spirochete numbers betweenthe wild-type and TLR1/2-deficient mice. Importantly, the present study identifies a cluster ofgenes regulated by TLR1/2-mediated signals. Future studies might reveal additional geneclusters altered by other TLRs and unfold “functional” gene clusters in the context of hostinnate immunity, helping to bridge the gap between the transcriptome and the functionalgenome of Borrelia. The molecular mechanisms that direct this change of expression are likelymultifactorial. Understanding the mechanisms by which these microbial changes are generatedmay lead to new strategies to combat infection with B. burgdorferi and other agents of disease.
AcknowledgmentsWe are grateful to Ira Schwartz for providing the Borrelia genome arrays, and we deeply appreciate his criticalscientific input during the preparation of the manuscript. We thank Kathleen DePonte and Deborah Beck for technicalassistance.
Financial support: National Institutes of Health (grants AI49200, AI32947, and AI053279). G.N. was supported byan Arthritis Foundation postdoctoral fellowship. E.F. and R.F. are investigators of the Howard Hughes MedicalInstitute.
References1. Steere AC, Coburn J, Glickstein L. The emergence of Lyme disease. J Clin Invest 2004;113:1093–
1101. [PubMed: 15085185]2. Steere AC, Glickstein L. Elucidation of Lyme arthritis. Nat Rev Immunol 2004;4:143–152. [PubMed:
15040587]3. Weis JJ. Host-pathogen interactions and the pathogenesis of murine Lyme disease. Curr Opin
Rheumatol 2002;14:399–403. [PubMed: 12118174]
Fikrig et al. Page 7
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
4. Rosa P. Microbiology of Borrelia burgdorferi. Semin Neurol 1997;17:5–10. [PubMed: 9166953]5. Casjens S, Palmer N, van Vugt R, et al. A bacterial genome in flux: the twelve linear and nine circular
extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borreliaburgdorferi. Mol Microbiol 2000;35:490–516. [PubMed: 10672174]
6. Fraser CM, Casjens S, Huang WM, et al. Genomic sequence of a Lyme disease spirochaete, Borreliaburgdorferi. Nature 1997;390:580–586. [PubMed: 9403685]
7. Rosa PA, Tilly K, Stewart PE. The burgeoning molecular genetics of the Lyme disease spirochaete.Nat Rev Microbiol 2005;3:129–143. [PubMed: 15685224]
8. Kabelitz D, Medzhitov R. Innate immunity—cross-talk with adaptive immunity through patternrecognition receptors and cytokines. Curr Opin Immunol 2007;19:1–3. [PubMed: 17157490]
9. Kawai T, Akira S. Antiviral signaling through pattern recognition receptors. J Biochem 2007;141:137–145. [PubMed: 17190786]
10. Kawai T, Akira S. TLR signaling. Semin Immunol 2007;19:24–32. [PubMed: 17275323]11. Alexopoulou L, Thomas V, Schnare M, et al. Hyporesponsiveness to vaccination with Borrelia
burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 2002;8:878–884.[PubMed: 12091878]
12. Hirschfeld M, Kirschning CJ, Schwandner R, et al. Cutting edge: inflammatory signaling by Borreliaburgdorferi lipoproteins is mediated by toll-like receptor 2. J Immunol 1999;163:2382–2386.[PubMed: 10452971]
13. Wooten RM, Ma Y, Yoder RA, et al. Toll-like receptor 2 is required for innate, but not acquired, hostdefense to Borrelia burgdorferi. J Immunol 2002;168:348–355. [PubMed: 11751980]
14. Wang G, Ma Y, Buyuk A, McClain S, Weis JJ, Schwartz I. Impaired host defense to infection andToll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficientC3H/HeJ mice. FEMS Microbiol Lett 2004;231:219–225. [PubMed: 14987768]
15. Wooten RM, Ma Y, Yoder RA, et al. Toll-like receptor 2 plays a pivotal role in host defense andinflammatory response to Borrelia burgdorferi. Vector Borne Zoonotic Dis 2002;2:275–278.[PubMed: 12804169]
16. Wang X, Ma Y, Yoder A, et al. T cell infiltration is associated with increased Lyme arthritis in TLR2−/− mice. FEMS Immunol Med Microbiol 2008;52:124–133. [PubMed: 18081848]
17. Bolz DD, Sundsbak RS, Ma Y, et al. MyD88 plays a unique role in host defense but not arthritisdevelopment in Lyme disease. J Immunol 2004;173:2003–2010. [PubMed: 15265935]
18. Liu N, Montgomery RR, Barthold SW, Bockenstedt LK. Myeloid differentiation antigen 88deficiency impairs pathogen clearance but does not alter inflammation in Borrelia burgdorferi-infected mice. Infect Immun 2004;72:3195–3203. [PubMed: 15155621]
19. Behera AK, Hildebrand E, Bronson RT, et al. MyD88 deficiency results in tissue-specific changesin cytokine induction and inflammation in interleukin-18-independent mice infected with Borreliaburgdorferi. Infect Immun 2006;74:1462–1470. [PubMed: 16495516]
20. Hovius JW, van Dam AP, Fikrig E. Tick-host-pathogen interactions in Lyme borreliosis. TrendsParasitol 2007;23:434–438. [PubMed: 17656156]
21. Rosa P. Lyme disease agent borrows a practical coat. Nat Med 2005;11:831–832. [PubMed:16079876]
22. Kraiczy P, Skerka C, Kirschfink M, Zipfel PF, Brade V. Immune evasion of Borrelia burgdorferi:insufficient killing of the pathogens by complement and antibody. Int J Med Microbiol2002;291:141–146. [PubMed: 12141738]
23. Cabello FC, Godfrey HP, Newman SA. Hidden in plain sight: Borrelia burgdorferi and theextracellular matrix. Trends Microbiol 2007;15:350–354. [PubMed: 17600717]
24. Liang FT, Nelson FK, Fikrig E. Molecular adaptation of Borrelia burgdorferi in the murine host. JExp Med 2002;196:275–280. [PubMed: 12119353]
25. Liang FT, Yan J, Mbow ML, et al. Borrelia burgdorferi changes its surface antigenic expression inresponse to host immune responses. Infect Immun 2004;72:5759–5767. [PubMed: 15385475]
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
27. Anguita J, Thomas V, Samanta S, et al. Borrelia burgdorferi-induced inflammation facilitatesspirochete adaptation and variable major protein-like sequence locus recombination. J Immunol2001;167:3383–3390. [PubMed: 11544329]
29. Neelakanta G, Li X, Pal U, et al. Outer surface protein B is critical for Borrelia burgdorferi adherenceand survival within Ixodes ticks. PLoS Pathog 2007;3:e33. [PubMed: 17352535]
30. Narasimhan S, Caimano MJ, Liang FT, et al. Borrelia burgdorferi transcriptome in the central nervoussystem of nonhuman primates. Proc Natl Acad Sci U S A 2003;100:15953–15958. [PubMed:14671329]
31. Ojaimi C, Brooks C, Casjens S, et al. Profiling of temperature-induced changes in Borreliaburgdorferi gene expression by using whole genome arrays. Infect Immun 2003;71:1689–1705.[PubMed: 12654782]
32. Narasimhan S, Sukumaran B, Bozdogan U, et al. A tick antioxidant facilitates the Lyme diseaseagent’s successful migration from the mammalian host to the arthropod vector. Cell Host Microbe2007;2:7–18. [PubMed: 18005713]
33. Parveen N, Cornell KA, Bono JL, Chamberland C, Rosa P, Leong JM. Bgp, a secretedglycosaminoglycan-binding protein of Borrelia burgdorferi strain N40, displays nucleosidaseactivity and is not essential for infection of immunodeficient mice. Infect Immun 2006;74:3016–3020. [PubMed: 16622242]
34. Shin OS, Isberg R, Akira S, Uematsu S, Behera AK, Hu LT. Distinct roles for MyD88, TLR2, TLR5,and TLR9 in phagocytosis of Borrelia burgdorferi and cytokine induction. Infect Immun2008;76:2341–2351. [PubMed: 18378636]
35. Liang FT, Jacobs MB, Bowers LC, Philipp MT. An immune evasion mechanism for spirochetalpersistence in Lyme borreliosis. J Exp Med 2002;195:415–422. [PubMed: 11854355]
36. Bernardino AL, Myers TA, Alvarez X, Hasegawa A, Philipp MT. Toll-like receptors: insights intotheir possible role in the pathogenesis of lyme neuroborreliosis. Infect Immun 2008;76:4385–4395.[PubMed: 18694963]
37. Eggers CH, Caimano MJ, Clawson ML, Miller WG, Samuels DS, Radolf JD. Identification of locicritical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the lyme disease spirochaete. MolMicrobiol 2002;43:281–295. [PubMed: 11985709]
38. Stewart PE, Chaconas G, Rosa P. Conservation of plasmidmaintenance functions between linear andcircular plasmids in Borrelia burgdorferi. J Bacteriol 2003;185:3202–3209. [PubMed: 12730180]
39. Purser JE, Lawrenz MB, Caimano MJ, Howell JK, Radolf JD, Norris SJ. A plasmid-encodednicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host. MolMicrobiol 2003;48:753–764. [PubMed: 12694619]
40. Schroder NW, Schombel U, Heine H, Gobel UB, Zahringer U, Schumann RR. Acylated cholesterylgalactoside as a novel immunogenic motif in Borrelia burgdorferi sensu stricto. J Biol Chem2003;278:33645–33653. [PubMed: 12810705]
41. Ben-Menachem G, Kubler-Kielb J, Coxon B, Yergey A, Schneerson R. A newly discoveredcholesteryl galactoside from Borrelia burgdorferi. Proc Natl Acad Sci U S A 2003;100:7913–7918.[PubMed: 12799465]
42. Yang Y, Li C. Transcription and genetic analyses of a putative N-acetylmuramyl-L-alanine amidasein Borrelia burgdorferi. FEMS Microbiol Lett 2009;290:164–173. [PubMed: 19025570]
43. Mulay V, Caimano MJ, Liveris D, Desrosiers DC, Radolf JD, Schwartz I. Borrelia burgdorferiBBA74, a periplasmic protein associated with the outer membrane, lacks porin-like properties. JBacteriol 2007;189:2063–2068. [PubMed: 17189354]
44. Revel AT, Talaat AM, Norgard MV. DNA microarray analysis of differential gene expression inBorrelia burgdorferi, the Lyme disease spirochete. Proc Natl Acad Sci U S A 2002;99:1562–1567.[PubMed: 11830671]
45. Brooks CS, Hefty PS, Jolliff SE, Akins DR. Global analysis of Borrelia burgdorferi genes regulatedby mammalian host-specific signals. Infect Immun 2003;71:3371–3383. [PubMed: 12761121]
Fikrig et al. Page 9
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
46. Tokarz R, Anderton JM, Katona LI, Benach JL. Combined effects of blood and temperature shift onBorrelia burgdorferi gene expression as determined by whole genome DNA array. Infect Immun2004;72:5419–5432. [PubMed: 15322040]
47. Mulay VB, Caimano MJ, Iyer R, et al. Borrelia burgdorferi bba74 is expressed exclusively duringtick feeding and is regulated by both arthropod and mammalian host-specific signals. J Bacteriol2009;191:2783–2794. [PubMed: 19218390]
48. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice:mutations in Tlr4 gene. Science 1998;282:2085–2088. [PubMed: 9851930]
49. Mysliwiec TH, Errington J, Vaidya AB, Bramucci MG. The Bacillus subtilis spo0J gene: evidencefor involvement in catabolite repression of sporulation. J Bacteriol 1991;173:1911–1919. [PubMed:1900505]
50. De Silva AM, Fikrig E. Growth and migration of Borrelia burgdorferi in Ixodes ticks during bloodfeeding. Am J Trop Med Hyg 1995;53:397–404. [PubMed: 7485694]
Fikrig et al. Page 10
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Figure 1.Microarray analysis of Borrelia burgdorferi gene expression in Toll-like receptor (TLR) 1 and2–deficient mouse skin. Shown is a representative autoradiogram image of the nylon arraysprobed with DECAL-enriched RNA from the skin of TLR1/2-deficient (A) and wild-type (B)mice infected with B. burgdorferi. Spots corresponding to bbe21 (1), bb0665 (2), bba74 (3),and bb0731 (4) are indicated.
Fikrig et al. Page 11
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Figure 2.Borrelia burgdorferi levels and selected gene expression in mice. A, Quantitative reverse-transcription polymerase chain reaction (qRTPCR) assessment of flaB levels relative to mouseβ-actin levels as a measure of viable Borrelia burden. B and C, qRT-PCR assessment ofbbe21, bb0665, bba74, and bb0731 transcripts in the skin (B) and bladders (C) of control andToll-like receptor (TLR) 1 and 2–deficient mice 2 weeks after infection. Asterisks (*) indicatea significant (P < .05, Student t test) increase in Borrelia burden, a significant increase inbbe21 and bb0665 expression, and a significant decrease in bb0731 and bba74 expression inTLR1/2-deficient mice compared with control mice. Data are means ± standard errors forvalues from 5–8 animals.
Fikrig et al. Page 12
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Figure 3.Borrelia burgdorferi lp25 levels and virulence in Toll-like receptor (TLR) 1 and 2–deficientmice. Shown are the results of quantitative polymerase chain reaction assessment of lp25 levelsin spirochetes from the bladders of wild-type and TLR1/2-deficient mice (A) and of flaB levelsas a measure of Borrelia burden in the skin and bladders of wild-type mice that received skingrafts from Borrelia-infected wild-type mice (wild type to wild type) and from TLR1/2-deficient mice (TLR1/2−/− to wild type) (B). Data are means ± standard errors for values from5 animals in each group of a representative experiment.
Fikrig et al. Page 13
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Figure 4.Borrelia burgdorferi bbe21, bb0665, bb0731, and bba74 expression in Ixodes scapularis.Shown are the results of quantitative reverse-transcription polymerase chain reactionassessment of flaB levels as a measure of viable Borrelia burden (A) and of bbe21 (B),bb0665 (C), bba74 (D), and bb0731 (E) transcripts in the unfed tick (unfed), in the midguts(MG) of ticks engorged on control (fed MG), in the salivary glands (SG) of ticks engorged oncontrol (fed SG), in the midguts of ticks engorged on Toll-like receptor (TLR) 1 and 2–deficientmice (fed MG TLR1/2−/−), and in the salivary glands of ticks engorged on TLR1/2-deficientmice (fed SG TLR1/2−/−). Asterisks (*) indicate a significant (P < .05, Student t test) increasein Borrelia burden and a significant decrease in bba74 and bb0731 expression in ticks fed onTLR1/2-deficient mice compared with those fed on control mice. Data are means ± standarderrors from a single experiment that was representative of duplicate experiments.
Fikrig et al. Page 14
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Fikrig et al. Page 15
Table 1
Borrelia burgdorferi Genes Expressed at Higher Levels in Toll-Like Receptor 1 and 2–Deficient Mice than inWild-Type Mice
Gene Function, homology, or category Cellular locationFold increase in
bb0585 Cell envelope/biosynthesis and degradation Cytoplasm 1.64
bb0715 Cell envelope/biosynthesis and degradation Cytoplasm 1.50
NOTE. Cellular locations were predicted using PSORTb software. Fold increases in expression were computed on the basis of the ratios of meanpixel intensity scores (normalized to flaB) assigned for each element by ImageJ software comparison of the nylon arrays and represent fold increasesin hybridization. Boldface type indicates genes analyzed in the present study.
aGene function derived from the literature.
bGene functions as annotated by the J. Craig Venter Institute (Maryland).
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
NIH
-PA Author Manuscript
Fikrig et al. Page 16
Table 2
Borrelia burgdorferi Genes Expressed at Higher Levels in Wild-Type Mice than in Toll-Like Receptor 1 and 2–Deficient Mice
Gene Function, homology, or category Cellular locationFold increase in
bb0795 Outer membrane protein (common protec-tive antigen)b
Outer membrane 2.56
bb0655 Heat-shock proteinb Cytoplasm 2.91
bb0074 Hypotheticalb No prediction 1.60
bb0520 Hypotheticalb No prediction 1.89
bb0560 Heat-shock protein 90b Cytoplasm 2.46
bb0012 tRNA pseudouridine synthase Cytoplasm 1.64
bb0443 spoIIIJ-associtated protein (jag) Cytoplasmic 1.93
bb0725 Hypothetical protein Cytoplasmic 1.72
NOTE. Cellular locations were predicted using PSORTb software. Fold increases in expression were computed on the basis of the ratios of meanpixel intensity scores (normalized to flaB) assigned for each element by ImageJ software comparison of the nylon arrays and represent fold increasesin hybridization. Boldface type indicates genes analyzed in the present study. tRNA, transfer RNA.
aGene function derived from the literature.
bGene functions annotated by the J. Craig Venter Institute (Maryland).
J Infect Dis. Author manuscript; available in PMC 2010 March 27.
