BACKGROUND: Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. RESULTS: Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain), and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. CONCLUSION: Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

Institute of Parasitology Biology Centre Academy of Sciences of the Czech Republic Ceské Budejovice CZ 370 05 The Czech Republic

Zobrazit více v PubMed

Hoogstraal H. Argasid and nuttalliellid ticks as parasites and vectors. Adv Parasitol. 1985;24:135–138. PubMed

Nutall PA. Pathogen-tick-host interactions: Borrelia bugdorferi and TBE virus. Zentralbl Bakteriol. 1999;289:492–505. PubMed

Grandjean O. Blood digestion in Ornithodoros moubata Murray sensu stricto Walton (Ixodoidea: Argasidae) females. I. Biochemical changes in the midgut lumen and ultrastructure of the midgut cells, related to intracellular digestion. Acarologia. 1984;25:147–165.

Sonenshine DE. Biology of Ticks. Vol. 1. New York: Oxford University Press; 1991.

Reich CI, Zorzopulos J. Boophilus microplus: characterization of larval proteases. Exp Parasitol. 1978;44:1–6. doi: 10.1016/0014-4894(78)90074-7. PubMed DOI

Coons LB, Rosell-Davis R, Tarnowski BI. Bloodmeal digestion in ticks. In: Sauer JR, Hair JA, editor. Morphology, Physiology, and Behavioral Biology of Ticks. Chichester, England: Ellis Horwood Ltd; 1986. pp. 248–279.

Coons LB, Alberti G. The Acari-Ticks. In: Harrison FW, Foelix R, editor. Microscopic Anatomy of Invertebrates, Chelicerate Arthropoda. 8B. NewYork: Wiley-Liss; 1999. pp. 267–514.

Lara FA, Lins U, Bechara GH, Oliveira PL. Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus. J Exp Biol. 2005;208:3093–3101. doi: 10.1242/jeb.01749. PubMed DOI

Lara FA, Lins U, Paiva-Silva G, Almeida IC, Braga CM, Miguens FC, Oliveira PL, Dansa-Petretski M. A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: aggregation inside a specialized organelle, the hemosome. J Exp Biol. 2003;206:1707–1715. doi: 10.1242/jeb.00334. PubMed DOI

Renard G, Garcia JF, Cardoso FC, Richter MF, Sakanari JA, Ozaki LS, Termignoni C, Masuda A. Cloning and functional expression of a Boophilus microplus cathepsin L-like enzyme. Insect Biochem Mol Biol. 2000;30:1017–1026. doi: 10.1016/S0965-1748(00)00070-9. PubMed DOI

Mulenga A, Sugimoto C, Ingram G, Ohashi K, Onuma M. Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes. J Vet Med Sci. 1999;61:497–503. doi: 10.1292/jvms.61.497. PubMed DOI

Boldbaatar D, Sikalizyo Sikasunge C, Battsetseg B, Xuan X, Fujisaki K. Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis. Insect Biochem Mol Biol. 2006;36:25–36. doi: 10.1016/j.ibmb.2005.10.003. PubMed DOI

Sojka D, Hajdušek O, Dvořák J, Sajid M, Franta Z, Schneider EL, Craik CS, Vancová M, Burešová V, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopáèek P. IrAE – An asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol. 2007;37:713–724. doi: 10.1016/j.ijpara.2006.12.020. PubMed DOI PMC

Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Motobu M, Fujisaki K. Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis. Insect Biochem Mol Biol. 2007;37:911–922. doi: 10.1016/j.ibmb.2007.04.010. PubMed DOI

Caffrey CR, McKerrow JH, Salter JP, Sajid M. Blood 'n' guts: an update on schistosome digestive peptidases. Trends Parasitol. 2004;20:241–248. doi: 10.1016/j.pt.2004.03.004. PubMed DOI

Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvořák J, Hsieh I, Bahgat M, Dissous C, McKerrow JH. A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem. 2006;281:39316–39329. doi: 10.1074/jbc.M607128200. PubMed DOI

Williamson AL, Brindley PJ, Knox DP, Hotez PJ, Loukas A. Digestive proteases of blood-feeding nematodes. Trends Parasitol. 2003;19:417–423. doi: 10.1016/S1471-4922(03)00189-2. PubMed DOI

Dalton JP, Clough KA, Jones MK, Brindley PJ. Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni. Infect Immun. 1996;64:1328–1334. PubMed PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. PubMed

SignalP 3.0 server

NetNGlyc 1.0 Server

Baig S, Damian RT, Peterson DS. A novel cathepsin B active site motif is shared by helminth bloodfeeders. Exp Parasitol. 2002;101:83–89. doi: 10.1016/S0014-4894(02)00105-4. PubMed DOI

Akov S. Blood digestion in ticks. In: Obenchain F, Galun R, editor. Physiology of Ticks. Oxford: Pergamon Press; 1982. pp. 197–211.

Mendiola J, Alonso M, Marquetti MC, Finlay C. Boophilus microplus: multiple proteolytic activities in the midgut. Exp Parasitol. 1996;82:27–33. doi: 10.1006/expr.1996.0004. PubMed DOI

Barrett AJ, Rawlings D, Woessner JF. Proteolytic enzymes. In: Oxford: Academic Press, editor. Handbook of Proteolytic Enzymes. Barrett AJ, Rawlings D, Woessner JF; 1998. pp. 801–805.

Sajid M, McKerrow JH, Hansell E, Mathieu MA, Lucas KD, Hsieh I, Greenbaum D, Bogyo M, Salter JP, Lim KC, Franklin C, Kim JH, Caffrey CR. Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase. Mol Biochem Parasitol. 2003;131:65–75. doi: 10.1016/S0166-6851(03)00194-4. PubMed DOI

Dvořák J, Delcroix M, Rossi A, Vopálenský V, Pospíšek M, Šedinová M, Mikeš L, Sajid M, Sali A, McKerrow JH, Horák P, Caffrey CR. Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition. Int J Parasitol. 2005;35:895–910. doi: 10.1016/j.ijpara.2005.02.018. PubMed DOI

Krupa JC, Hasnain S, Nagler DK, Menard R, Mort JS. S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B. Biochem J. 2002;361:613–619. doi: 10.1042/0264-6021:3610613. PubMed DOI PMC

Pham CTN, Ley TJ. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc Nat Acad Sci. 1999;96:8627–8632. doi: 10.1073/pnas.96.15.8627. PubMed DOI PMC

Miyoshi T, Tsuji N, Islam MK, Huang X, Motobu M, Alim MA, Fujisaki K. Molecular and reverse genetic characterization of serine proteinase-induced hemolysis in the midgut of the ixodid tick Haemaphysalis longicornis. J Insect Physiol. 2007;53:195–203. doi: 10.1016/j.jinsphys.2006.12.001. PubMed DOI

Logullo C, Vaz IS, Sorgine MH, Paiva-Silva GO, Faria FS, Zingali RB, De Lima MF, Abreu L, Oliveira EF, Alves EW, Masuda H, Gonzales JC, Masuda A, Oliveira PL. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus. Parasitology. 1998;116:525–532. doi: 10.1017/S0031182098002698. PubMed DOI

Sorgine MH, Logullo C, Zingali RB, Paiva-Silva GO, Juliano L, Oliveira PL. A heme-binding aspartic proteinase from the eggs of the hard tick Boophilus microplus. Biol Chem. 2000;275:28659–28665. doi: 10.1074/jbc.M005675200. PubMed DOI

Hill CA, Wikel SK. The Ixodes scapularis Genome Project: an opportunity for advancing tick research. Trends Parasitol. 2005;21:151–153. doi: 10.1016/j.pt.2005.02.004. PubMed DOI

Tort J, Brindley PJ, Knox D, Wolfe KH, Dalton JP. Proteinases and associated genes of parasitic helminths. Adv Parasitol. 1999;43:161–266. PubMed

Oliver EM, Skuce PJ, McNair CM, Knox DP. Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus. Parasitology. 2006;133:237–244. doi: 10.1017/S0031182006000229. PubMed DOI

Rosenthal PJ. Cysteine proteases of malaria parasites. Int J Parasitol. 2004;34:1489–1499. doi: 10.1016/j.ijpara.2004.10.003. PubMed DOI

Liu J, Istvan ES, Gluzman IY, Gross J, Goldberg DE. Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems. Proc Nat Acad Sci. 2006;103:8840–8805. doi: 10.1073/pnas.0601876103. PubMed DOI PMC

de la Fuente J, Kocan KM. Strategies for development of vaccines for control of ixodid tick species. Parasite Immunol. 2006;28:275–283. doi: 10.1111/j.1365-3024.2006.00828.x. PubMed DOI

Partanen S, Storch S, Löffler HG, Hasilik A, Tyynelä J, Braulke T. A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells. Biochem J. 2003;369:55–62. doi: 10.1042/BJ20021226. PubMed DOI PMC

Máša M, Marešová L, Vondrášek JR, Horn M, Ježek J, Mareš M. Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core. Biochemistry. 2006;45:15474–15482. doi: 10.1021/bi0614986. PubMed DOI

Green GD, Shaw E. Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. J Biol Chem. 1981;256:1923–1928. PubMed

Ekici ÖD, Götz MG, James KE, Li ZZ, Rukamp BJ, Asgian JL, Caffrey CR, Hansell E, Dvořák J, McKerrow JH, Potempa J, Travis J, Mikolajczyk J, Salvesen GS, Powers JC. Aza-peptide Michael acceptors: a new class of inhibitors specific for caspases and other clan CD cysteine proteases. J Med Chem. 2004;47:1889–1892. doi: 10.1021/jm049938j. PubMed DOI

Demoz M, Castino R, Follo C, Hasilik A, Sloane BF, Isidoro C. High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells. Protein Expr Purif. 2006;45:157–167. doi: 10.1016/j.pep.2005.07.024. PubMed DOI

Barrett AJ, Kirschke H. Cathepsin B, Cathepsin H, and Cathepsin L. Methods Enzymol. 1981;80:535–561. PubMed

McGuire MJ, Lipsky PE, Thiele DL. Purification and characterization of dipeptidyl peptidase I from human spleen. Arch Biochem Biophys. 1992;295:280–288. doi: 10.1016/0003-9861(92)90519-3. PubMed DOI

Kembhavi AA, Buttle DJ, Knight CG, Barrett AJ. The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays. Arch Biochem Biophys. 1993;303:208–213. doi: 10.1006/abbi.1993.1274. PubMed DOI

Grunclová L, Horn M, Vancová M, Sojka D, Franta Z, Mareš M, Kopáček P. Two secreted cystatins of the soft tick Ornithodoros moubata: differential expression pattern and inhibitory specificity. Biol Chem. 2006;387:1635–1644. doi: 10.1515/BC.2006.204. PubMed DOI

Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, Towatari T, Nikawa T, Katunum N. Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. FEBS Lett. 1991;25:307–310. doi: 10.1016/0014-5793(91)80318-W. PubMed DOI

Baricos WH, Zhou Y, Mason RW, Barrett AJ. Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane. Biochem J. 1988;252:301–344. PubMed PMC

Knight CG, Barrett AJ. Interaction of human cathepsin D with the inhibitor pepstatin. Biochem J. 1976;155:117–125. PubMed PMC

blastn

Rego RO, Kovář V, Kopáček P, Weise C, Man P, Šauman I, Grubhoffer L. The tick plasma lectin, Dorin M, is a fibrinogen-related molecule. Insect Biochem Mol Biol. 2006;36:291–299. doi: 10.1016/j.ibmb.2006.01.008. PubMed DOI

MEROPS database

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876–4882. doi: 10.1093/nar/25.24.4876. PubMed DOI PMC

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95–98.

Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406–425. PubMed

Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 2001;17:1244–1255. doi: 10.1093/bioinformatics/17.12.1244. PubMed DOI

Kopáček P, Ždychová J, Yoshiga T, Weise C, Rudenko N, Law JH. Molecular cloning, expression and isolation of ferritins from two tick species–Ornithodoros moubata and Ixodes ricinus. Insect Biochem Mol Biol. 2003;233:103–113. doi: 10.1016/S0965-1748(02)00181-9. PubMed DOI

Insight Into the Dynamics of the Ixodes ricinus Nymphal Midgut Proteome

Blood-feeding adaptations and virome assessment of the poultry red mite Dermanyssus gallinae guided by RNA-seq

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion

RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks

Interaction of the tick immune system with transmitted pathogens

Characterization of gut-associated cathepsin D hemoglobinase from tick Ixodes ricinus (IrCD1)

Cysteine proteases from bloodfeeding arthropod ectoparasites

Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

Hemoglobin digestion in blood-feeding ticks: mapping a multipeptidase pathway by functional proteomics

Knockdown of proteins involved in iron metabolism limits tick reproduction and development