Reverse genetic approach to assess function of molecules involved in the tick innate immunity, pathogen transmission and blood digestion of the hard tick Ixodes ricinus. Development of anti-tick vaccines.
Chmelař J., Oliveira C.J., Řezáčová P., Francischetti I.M., Kovářová Z., Pejler G., Kopáček P., Ribeiro J.M., Mareš M., Kopecký J., Kotsyfakis M. (2011) A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood 117: 736–744.
Franta Z., Sojka D., Frantová H., Dvořák J., Horn M., Srba J., Talacko P., Mareš M., Schneider E., Craik C.S., McKerrow J.H., Caffrey C.R., Kopáček P. (2011) IrCL1-The haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus. International Journal for Parasitology 41: 1253–1262.
Hajdušek O., Almazán C., Loosová G., Villar M., Canales M., Grubhoffer L., Kopáček P., de la Fuente J. (2010) Characterization of ferritin 2 for the control of tick infestations. Vaccine 28: 2993–2998.
Franta Z., Frantová H., Konvičková J., Horn M., Sojka D., Mareš M., Kopáček P. (2010) Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus. Parasites & Vectors 3: 119.
HAJDUŠEK O., SOJKA D., KOPÁČEK P., BUREŠOVÁ V., FRANTA Z., ŠAUMAN I., WINZERLING J., GRUBHOFFER L. (2009) Knockdown of proteins involved in iron metabolism limits tick reproduction and development. Proceedings of the National Academy of Sciences 109: 1033-1038.
Pathogens transmitted by a blood-sucking disease vector have to be capable of withstanding the innate defense system of the invertebrate host, which is comprised of the cellular and humoral immune responses. In contrast to blood-sucking insects, our understanding of innate immunity in ticks is still very scanty. Thus far, only a limited number of cellular immune reactions (phagocytosis, nodulation, encapsulation) and immune molecules (antimicrobial peptides, lysozymes, lectins) have been described in ticks (for review see, Kopáček et al., 2010 ). Originally we used the African soft tick Ornithodoros moubata as a model tick for classical (protein to gene) characterization of tick immune molecules. The first molecule we isolated from the gut contents of this species was lysozyme – a defense protein active against Gram+ bacteria (Kopáček et al., 1999). Its sequence displays features of lysozymes with antibacterial as well as digestive function and the gene is significantly up-regulated upon blood-meal (Grunclová et al., 2003). A sialic–acid specific lectin named DorinM was purified from O. moubata hemolymph (Kovář et al., 2000) and its sequencing revealed that it is a fibrinogen-related protein (Rego et al., 2006) similar to the horseshoe crab tachylectins 5A and5B playing a role as a pattern recognition molecules in the initial stage of the immune response. Another abundant glycoprotein from O. moubata plasma was isolated and identified as a pan-protease inhibitor of ?2-macroglobulin (?2M) type, the first ?2M described in a terrestrial invertebrate (Kopáček et al., 2000). We have cloned and sequenced the TAM (for tick alpha-2-macroglobulin) and demonstrated that this large molecule displays quite atypical structural features (Saravanan et al. 2003). Our present approach in the research of tick immunity relies mainly on the reverse genetics and is preferentially focused on the hard tick Ixodes ricinus, the most serious arthropod vector of Lyme disease and tick borne encephalitis in Europe. The introducing of RNA interference in our laboratory made it possible to perform a functional study of ?2M from the hard tick I. ricinus (IrAM) revealing that this protein is involved in phagocytosis of a potential pathogen Chryseobacterium indologenes by tick hemocytes (Burešová et al., 2006; Burešová et al., 2009). Our recent genome-wide screening of the related Ixodes scapularis and functional genomics of corresponding orthologs in I. ricinus revealed, that these hard ticks possess representatives of all major groups of invertebrate thioester-protein family, namely ?2-macroglobulins, complement C3-related proteins, insect TEPs and macroglobulin-complement-related molecules. This finding makes ticks an exceptional organism for further study of primitive complement-like system, since the presence of all groups of thioester-proteins has not yet been described in any other organism.
Mapping of hemoglobinolytic peptidases in the gut of the tick Ixodes ricinus
Ticks are vectors for a variety of viral, bacterial and parasitic disease in human and domestic animals. As obligate blood-feeders, one possible strategy to control ticks or retard disease transmission is to impair parasite’s ability to digest host proteins. Our research on tick digestive machinery is performed in tight cooperation with Peptidase laboratory of Dr. Michael Mareš at the Institute of Organic Chemistry and Biochemistry, ASCR, Prague and the Laboratory of Prof. James McKerrow and Conor R. Caffrey at the Sandler Centre for Basic Research in Parasitic Diseases, UCSF, San Francisco. We have started the research in this field working on the model soft tick Ornithodoros moubata, in which we have characterized the cysteine protease inhibitors of cystatin type (Grunclová et al., 2006, Salát et al., 2010). At present, we are focused on the hard tick Ixodes ricinus, the relevant European vector of tick-borne encephalitis and Lyme disease. We subsequently characterize the main players involved in the blood digestion, e.g. the asparaginyl endopeptidase, the first legumain described in arthropods (Sojka et al., 2007). In addition, we use the three-pronged strategy including reverse genetic approach, immunohistochemistry and selective inhibitor based proteomic analysis of the gut extract to decipher the entire peptidase network involved in the blood digestion in the tick gut cells (Sojka et al., 2008; Horn et al., 2009). The digestive network comprising a successive action of aspartic and cysteine peptidases displays a remarkable similarity with distant hematophagous parasites such as nematodes and flatworms. This indicates that cystein/aspartic peptidase system for blood digestion is evolutionarily conserved but it differs substantially from blood-sucking insects relying mainly on serine peptidases. An improved insight in the tick digestion promises to discover the most vulnerable ‘Achilles heels’ in the process which may in further lead to candidates for an efficient ‘anti-tick’ vaccine.
The interest in how ticks manages the surplus of iron originating from the host blood started during Petr Kopáček’s stay in the Laboratory of Prof. John H. Law, at University of Arizona, Tucson in 1997. The focus on tick iron metabolism resulted in description of tick ferritins (FER1) which are quite similar to the vertebrate heavy-chain type ferritins functioning as intracellular iron storage molecules (Kopáček et al., 2003). The further functional research of tick iron metabolism employing the method of RNA interference led to the characterization of the iron-regulatory protein’ (IRP) and more importantly, to the discovery of a novel type of ferritin (FER2), which is secreted into the tick plasma where it serves as iron transporter (Hajdušek et al., 2009). Elimination of ferritin 2 by RNAi severely affected tick ability to feed on the host. A similar effect was achieved by experimental vaccination of rabbits with recombinant ferritin 2, where the antibodies imbibed with the host blood efficiently blocked ferritin 2 in the tick gut. This fact, together with a remarkable molecular difference from ferritins of mammalian hosts make the tick ferritin 2 a promising candidate for an efficient ‘anti-tick’ vaccine protecting from tick feeding and limiting their ability to transmit pathogens causing infection diseases. The use of ferritin 2 as a veterinary vaccine is protected by a Czech and an international patent claims. The potential of the vaccine based on the recombinant ferritin 2 for an efficient tick control is currently investigated and the tests on the cattle brings first promising results (Hajdušek et al., 2010).
A large collection of helminths is available for comparative studies...
