Laboratory of Molecular Biology of Ticks
Research in our laboratory is focused on molecular descriptions of proteins that are key for the successful blood-feeding of ticks (mainly Ixodes ricinus) and the red poultry mite (Dermanyssus gallinae), or play a role in the acquisition & transmission of tick-borne pathogens. We cover a wide range of topics including metal biology, description of proteolytic apparatuses, systemic and epithelial immunity to experimental infection, integration of functional microbiota, and mapping of protein-ligand interactions.
Our keystone experimental approaches comprise:
i) ex vivo artificial membrane feeding for all developmental stages of I. ricinus ticks and adults of D. gallinae mites
ii) RNA interference in Ixodes spp. ticks or D. gallinae mites
iii) An acquisition & transmission mouse model for Ixodes ricinus - Borrelia afzelii
iv) Vaccination studies against ticks, D. gallinae mites, and Borrelia transmission
v) A culture model for Babesia divergens and an animal model for Babesia microti
vi) A culture model for Anaplasma phagocytophilum
vii) Recombinant protein expression & purification (enzyme kinetics, binding assays, vaccination studies)
viii) Immunodetection of proteins in tick tissues using confocal microscopy, TEM, and array tomography
Apart from basic research, we also carry out contractual research offering throughput ex vivo membrane feeding for acaricidal screening through oral ingestion of blood meal, or in vivo vaccine assessment studies against ticks or D. gallinae mites in small mammals or hens respectively.
Tick Innate Immunity
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 alpha2-macroglobulin (alpha2M) type, the first alpha2M 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 alpha2M 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 alpha2-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.
Tick gut, iron metabolism and blood digestion
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.