Ixodes ricinus is the vector for Borrelia afzelii, the predominant cause of Lyme borreliosis in Europe, whereas Ixodes scapularis is the vector for Borrelia burgdorferi in the USA. Transcription of several I. scapularis genes changes in the presence of B. burgdorferi and contributes to successful infection. To what extend B. afzelii influences gene expression in I. ricinus salivary glands is largely unknown. Therefore, we measured expression of uninfected vs. infected tick salivary gland genes during tick feeding using Massive Analysis of cDNA Ends (MACE) and RNAseq, quantifying 26.179 unique transcripts. While tick feeding was the main differentiator, B. afzelii infection significantly affected expression of hundreds of transcripts, including 465 transcripts after 24 h of tick feeding. Validation of the top-20 B. afzelii-upregulated transcripts at 24 h of tick feeding in ten biological genetic distinct replicates showed that expression varied extensively. Three transcripts could be validated, a basic tail protein, a lipocalin and an ixodegrin, and might be involved in B. afzelii transmission. However, vaccination with recombinant forms of these proteins only marginally altered B. afzelii infection in I. ricinus-challenged mice for one of the proteins. Collectively, our data show that identification of tick salivary genes upregulated in the presence of pathogens could serve to identify potential pathogen-blocking vaccine candidates.

Biology Centre Institute of Parasitology Czech Academy of Sciences Ceske Budejovice Czech Republic

Center for Experimental and Molecular Medicine Amsterdam Infection and Immunity Amsterdam UMC Location Academic Medical Center University of Amsterdam Amsterdam The Netherlands

CIC bioGUNE Basque Research and Technology Alliance 48160 Derio Spain

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

GenXPro GmbH Frankfurt Innovation Center Biotechnology Frankfurt am Main Germany

Ikerbasque Basque Foundation for Science 48012 Bilbao Spain

National Institute for Public Health and the Environment Bilthoven The Netherlands

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Eisen RJ, Eisen L. The blacklegged tick, Ixodes scapularis: an increasing public health concern. Trends Parasitol. 2018;34:295–309. doi: 10.1016/j.pt.2017.12.006. PubMed DOI PMC

Semenza JC, Suk JE. Vector-borne diseases and climate change: a European perspective. FEMS Microbiol. Lett. 2017 doi: 10.1093/femsle/fnx244. PubMed DOI PMC

Hubálek Z. Epidemiology of lyme borreliosis. Curr. Probl. Dermatol. 2009;37:31–50. doi: 10.1159/000213069. PubMed DOI

van Dam AP, et al. Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin .Infect. Dis. 1993;17:708–717. doi: 10.1093/clinids/17.4.708. PubMed DOI

Embers ME, Narasimhan S. Vaccination against Lyme disease: past, present, and future. Front. Cell. Infect. Microbiol. 2013;3:6–6. doi: 10.3389/fcimb.2013.00006. PubMed DOI PMC

Cook MJ. Lyme borreliosis: a review of data on transmission time after tick attachment. Int. J. Gen. Med. 2014;8:1–8. doi: 10.2147/IJGM.S73791. PubMed DOI PMC

Crippa M, Rais O, Gern L. Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks. Vector Borne Zoonotic Dis. 2002;2:3–9. doi: 10.1089/153036602760260724. PubMed DOI

Pospisilova T, et al. Tracking Borrelia afzelii from infected Ixodes ricinus nymphs to mice suggests a direct 'gut-to-mouth' route of Lyme disease transmission. J. bioRxiv. 2018 doi: 10.1101/316927. DOI

Narasimhan S, et al. Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance. Ticks Tick Borne Dis. 2020;101:369. doi: 10.1016/j.ttbdis.2019.101369. PubMed DOI PMC

Schuijt TJ, et al. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display. PLoS ONE. 2011;6:e15926. doi: 10.1371/journal.pone.0015926. PubMed DOI PMC

Trager W. Acquired immunity to ticks. J. Parasitol. 1939;25:57–81. doi: 10.2307/3272160. DOI

Anguita J, et al. Salp15, an Ixodes scapularis salivary protein, inhibits CD4(+) T cell activation. Immunity. 2002;16:849–859. doi: 10.1016/S1074-7613(02)00325-4. PubMed DOI

Dai J, et al. Tick histamine release factor is critical for Ixodes scapularis engorgement and transmission of the lyme disease agent. PLoS Pathog. 2010;6:e1001205. doi: 10.1371/journal.ppat.1001205. PubMed DOI PMC

Dai J, et al. Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent. Cell Host Microbe. 2009;6:482–492. doi: 10.1016/j.chom.2009.10.006. PubMed DOI PMC

Schuijt TJ, et al. A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the lyme disease agent. Cell Host Microbe. 2011;10:136–146. doi: 10.1016/j.chom.2011.06.010. PubMed DOI PMC

Wagemakers, A. et al. An Ixodes ricinus tick salivary lectin pathway inhibitor protects Borrelia burgdorferi sensu lato from human complement (2016). PubMed

Brossard M, Girardin P. Passive transfer of resistance in rabbits infested with adult Ixodes ricinus L: humoral factors influence feeding and egg laying. Experientia. 1979;35:1395–1397. doi: 10.1007/BF01964030. PubMed DOI

Narasimhan S, et al. Immunity against Ixodes scapularis salivary proteins expressed within 24 hours of attachment thwarts tick feeding and impairs Borrelia transmission. PLoS ONE. 2007;2:e451. doi: 10.1371/journal.pone.0000451. PubMed DOI PMC

Nazario S, et al. Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity. Am. J. Trop. Med. Hygiene. 1998;58:780–785. doi: 10.4269/ajtmh.1998.58.780. PubMed DOI

Wikel SK, Ramachandra RN, Bergman DK, Burkot TR, Piesman J. Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infect. Immun. 1997;65:335–338. doi: 10.1128/IAI.65.1.335-338.1997. PubMed DOI PMC

Chmelar J, et al. Insight into the sialome of the castor bean tick, Ixodes ricinus. BMC Genom. 2008;9:233. doi: 10.1186/1471-2164-9-233. PubMed DOI PMC

Perner J, Kropáčková S, Kopáček P, Ribeiro JMC. Sialome diversity of ticks revealed by RNAseq of single tick salivary glands. PLOS Negl. Trop. Dis. 2018;12:e0006410. doi: 10.1371/journal.pntd.0006410. PubMed DOI PMC

Schwarz A, et al. De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies. FASEB J. 2013;27:4745–4756. doi: 10.1096/fj.13-232140. PubMed DOI PMC

Kotsyfakis M, Schwarz A, Erhart J, Ribeiro JMC. Tissue- and time-dependent transcription in Ixodes ricinus salivary glands and midguts when blood feeding on the vertebrate host. Sci. Rep. 2015;5:9103. doi: 10.1038/srep09103. PubMed DOI PMC

Anguita, J. et al. Salp15, an ixodes scapularis salivary protein, inhibits CD4(+) T cell activation. PubMed

Cotte V, et al. Differential expression of Ixodes ricinus salivary gland proteins in the presence of the Borrelia burgdorferi sensu lato complex. J. Proteomics. 2014;96:29–43. doi: 10.1016/j.jprot.2013.10.033. PubMed DOI

Hovius JW, et al. Preferential protection of Borrelia burgdorferi sensu stricto by a salp 15 homologue in Ixodes ricinus saliva. J. Infect. Dis. 2008;198:1189–1197. doi: 10.1086/591917. PubMed DOI PMC

Hovius JW, van Dam AP, Fikrig E. Tick–host–pathogen interactions in Lyme borreliosis. Trends Parasitol. 2007;23:434–438. doi: 10.1016/j.pt.2007.07.001. PubMed DOI

Narasimhan S, et al. A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission. PLoS Pathog. 2014;10:e1004278. doi: 10.1371/journal.ppat.1004278. PubMed DOI PMC

Francischetti IM, Sa-Nunes A, Mans BJ, Santos IM, Ribeiro JM. The role of saliva in tick feeding. Front. Biosci. (Landmark edition) 2009;14:2051–2088. doi: 10.2741/3363. PubMed DOI PMC

Mans BJ, Featherston J, de Castro MH, Pienaar R. Gene duplication and protein evolution in tick–host interactions. Front. Cell. Infect. Microbiol. 2017;7:413. doi: 10.3389/fcimb.2017.00413. PubMed DOI PMC

Nold-Petry CA, et al. IL-37 requires the receptors IL-18Ralpha and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction. Nat. Immunol. 2015;16:354–365. doi: 10.1038/ni.3103. PubMed DOI

Zawada AM, et al. Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease. Epigenetics. 2014;9:161–172. doi: 10.4161/epi.26931. PubMed DOI PMC

Mandelboum S, Manber Z, Elroy-Stein O, Elkon R. Recurrent functional misinterpretation of RNA-seq data caused by sample-specific gene length bias. PLoS Biol. 2019;17:e3000481. doi: 10.1371/journal.pbio.3000481. PubMed DOI PMC

Asmann YW, et al. 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer. BMC Genom. 2009;10:531. doi: 10.1186/1471-2164-10-531. PubMed DOI PMC

Lenz TL, Eizaguirre C, Rotter B, Kalbe M, Milinski M. Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide digital gene expression analysis. Mol. Ecol. 2013;22:774–786. doi: 10.1111/j.1365-294X.2012.05756.x. PubMed DOI PMC

Kotsyfakis M, Schwarz A, Erhart J, Ribeiro JMC. Tissue- and time-dependent transcription in Ixodes ricinus salivary glands and midguts when blood feeding on the vertebrate host. Sci. Rep. 2015;5:1. doi: 10.1038/srep09103. PubMed DOI PMC

Schwarz A, et al. A systems level analysis reveals transcriptomic and proteomic complexity in Ixodes ricinus midgut and salivary glands during early attachment and feeding. Mol. Cell. Proteomics. 2014;13:2725–2735. doi: 10.1074/mcp.M114.039289. PubMed DOI PMC

Schwarz A, et al. De novo Ixodes ricinus salivary gland transcriptome analysis using two next-generation sequencing methodologies. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2013;27:4745–4756. doi: 10.1096/fj.13-232140. PubMed DOI PMC

Piesman J, Happ CM. The efficacy of co-feeding as a means of maintaining Borrelia burgdorferi: a North American model system. J. Vector Ecol. 2001;26:216–220. PubMed

Pospisilova T, et al. Tracking of Borrelia afzelii transmission from infected Ixodes ricinus nymphs to mice. Infect. Immun. 2019 doi: 10.1128/IAI.00896-18. PubMed DOI PMC

Crippa M, Rais O, Gern L. Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks. Vector Borne Zoonotic Dis. (Larchmont, N.Y.) 2002;2:3–9. doi: 10.1089/153036602760260724. PubMed DOI

Piesman J, Mather TN, Sinsky RJ, Spielman A. Duration of tick attachment and Borrelia burgdorferi transmission. J. Clin. Microbiol. 1987;25:557–558. doi: 10.1128/JCM.25.3.557-558.1987. PubMed DOI PMC

Chmelar J, Kotal J, Kovarikova A, Kotsyfakis M. The use of tick salivary proteins as novel therapeutics. Front. Physiol. 2019;10:812. doi: 10.3389/fphys.2019.00812. PubMed DOI PMC

Assumpcao TCF, Ribeiro JMC, Francischetti IMB. Disintegrins from hematophagous sources. Toxins (Basel) 2012;4:296–322. doi: 10.3390/toxins4050296. PubMed DOI PMC

Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. Nat. Rev. Mol. Cell Biol. 2002;3:932–943. doi: 10.1038/nrm977. PubMed DOI

Garcia RC, Murgia R, Cinco M. Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner. Infect. Immun. 2005;73:6138. doi: 10.1128/IAI.73.9.6138-6142.2005. PubMed DOI PMC

Carreras-González A, et al. Regulation of macrophage activity by surface receptors contained within Borrelia burgdorferi-enriched phagosomal fractions. PLoS Pathog. 2019;15:e1008163. doi: 10.1371/journal.ppat.1008163. PubMed DOI PMC

Hawley KL, et al. CD14 cooperates with complement receptor 3 to mediate MyD88-independent phagocytosis of Borrelia burgdorferi. Proc. Natl. Acad. Sci. U. S. A. 2012;109:1228–1232. doi: 10.1073/pnas.1112078109. PubMed DOI PMC

Daix V, et al. Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins. Insect. Mol. Biol. 2007;16:155–166. doi: 10.1111/j.1365-2583.2006.00710.x. PubMed DOI

Hourcade DE, et al. Anti-complement activity of the Ixodes scapularis salivary protein Salp20. Mol. Immunol. 2016;69:62–69. doi: 10.1016/j.molimm.2015.11.008. PubMed DOI PMC

Valenzuela JG, Charlab R, Mather TN, Ribeiro JM. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. J. Biol. Chem. 2000;275:18717–18723. doi: 10.1074/jbc.M001486200. PubMed DOI

Coumou J, et al. The role of mannose binding lectin in the immune response against Borrelia burgdorferi sensu lato. Sci. Rep. 2019;9:1431. doi: 10.1038/s41598-018-37922-8. PubMed DOI PMC

Hawley KL, Olson CM, Jr, Carreras-González A, Navasa N, Anguita J. Serum C3 enhances complement receptor 3-mediated phagocytosis of Borrelia burgdorferi. Int. J. Biol. Sci. 2015;11:1269–1271. doi: 10.7150/ijbs.13395. PubMed DOI PMC

Bowessidjaou J, Brossard M, Aeschlimann A. Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus L. Experientia. 1977;33:528–530. doi: 10.1007/BF01922254. PubMed DOI

Hovius JWR, et al. The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi. PLoS Pathog. 2009;5:e1000447–e1000447. doi: 10.1371/journal.ppat.1000447. PubMed DOI PMC

Tang J, et al. YY-39, a tick anti-thrombosis peptide containing RGD domain. Peptides. 2015;68:99–104. doi: 10.1016/j.peptides.2014.08.008. PubMed DOI

Andersen JF, Gudderra NP, Francischetti IM, Valenzuela JG, Ribeiro JM. Recognition of anionic phospholipid membranes by an antihemostatic protein from a blood-feeding insect. Biochemistry. 2004;43:6987–6994. doi: 10.1021/bi049655t. PubMed DOI PMC

Das S, et al. Salp25D, an Ixodes scapularis antioxidant, is 1 of 14 immunodominant antigens in engorged tick salivary glands. J. Infect. Dis. 2001;184:1056–1064. doi: 10.1086/323351. PubMed DOI

Narasimhan S, et al. A novel family of anticoagulants from the saliva of Ixodes scapularis. Insect. Mol. Biol. 2002;11:641–650. doi: 10.1046/j.1365-2583.2002.00375.x. PubMed DOI

Narasimhan S, et al. Disruption of Ixodes scapularis anticoagulation by using RNA interference. Proc. Natl. Acad. Sci. U. S. A. 2004;101:1141–1146. doi: 10.1073/pnas.0307669100. PubMed DOI PMC

Aase A, et al. Validate or falsify: lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood. Infect. Dis. (Lond.) 2016;48:411–419. doi: 10.3109/23744235.2016.1144931. PubMed DOI

de la Fuente J, et al. Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Vet. Parasitol. 2005;133:357–362. doi: 10.1016/j.vetpar.2005.05.063. PubMed DOI

Parkhomchuk D, et al. Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res. 2009;37:e123–e123. doi: 10.1093/nar/gkp596. PubMed DOI PMC

Haas BJ, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 2013;8:1494. doi: 10.1038/nprot.2013.084. PubMed DOI PMC

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. 2011;17(3):2011. doi: 10.14806/ej.17.1.200. DOI

Consortium, T. U UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2018;47:D506–D515. doi: 10.1093/nar/gky1049. PubMed DOI PMC

Gasteiger E, et al. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2003;31:3784–3788. doi: 10.1093/nar/gkg563. PubMed DOI PMC

Mitchell AL, et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2019;47:D351–D360. doi: 10.1093/nar/gky1100. PubMed DOI PMC

Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 2015;10:845–858. doi: 10.1038/nprot.2015.053. PubMed DOI PMC

Almagro Armenteros JJ, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019;37:420–423. doi: 10.1038/s41587-019-0036-z. PubMed DOI

Steentoft C, et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J. 2013;32:1478–1488. doi: 10.1038/emboj.2013.79. PubMed DOI PMC

Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins. 2004;46:203–206.

Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes 11 Edited by F. Cohen. J. Mol. Biol. 2001;305:567–580. doi: 10.1006/jmbi.2000.4315. PubMed DOI

Fankhauser N, Mäser P. Identification of GPI anchor attachment signals by a Kohonen self-organizing map. Bioinformatics. 2005;21:1846–1852. doi: 10.1093/bioinformatics/bti299. PubMed DOI

Reynisson B, Alvarez B, Paul S, Peters B, Nielsen M. NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res. 2020 doi: 10.1093/nar/gkaa379. PubMed DOI PMC

Jespersen MC, Peters B, Nielsen M, Marcatili P. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 2017;45:W24–W29. doi: 10.1093/nar/gkx346. PubMed DOI PMC

Sheffield P, Garrard S, Derewenda Z. Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors. Protein Expr. Purif. 1999;15:34–39. doi: 10.1006/prep.1998.1003. PubMed DOI

Schwaiger M, Péter O, Cassinotti P. Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay. Clin. Microbiol. Infect. 2001;7:461–469. doi: 10.1046/j.1198-743x.2001.00282.x. PubMed DOI

Activation of the tick Toll pathway to control infection of Ixodes ricinus by the apicomplexan parasite Babesia microti

Probing an Ixodes ricinus salivary gland yeast surface display with tick-exposed human sera to identify novel candidates for an anti-tick vaccine

Identification of Tick Ixodes ricinus Midgut Genes Differentially Expressed During the Transmission of Borrelia afzelii Spirochetes Using a Transcriptomic Approach