Laboratory of genomics and proteomics of disease vectors (M. Kotsyfakis)
The research activity of our group focuses on:
- Applying genomic and proteomic methodologies to discover salivary or midgut effectors that determine arthropod vectorial capacity.
- Vertebrate proteolytic cascades that are regulated by arthropod salivary secretion in the sites of disease vector infestation, facilitating blood meal uptake by the vector and/or pathogen transmission.
- Demonstrating the pharmacological action of arthropod salivary proteins in the vertebrate host (hemostasis, vascular biology, immunomodulation) and their potential for the treatment of human diseases (cancer, multiple sclerosis, allergic asthma) using transgenic animals and animal models for human diseases.
The research projects of our group aim:
- To explore the basic mechanisms of pathogen transmission by disease vectors as the conceptual basis for the development of novel methods/tools (e.g. vaccines, diagnostic kits) for controlling vector-borne diseases.
- To test recombinant arthropod salivary proteins in animal models (for human diseases) with an ultimate goal to describe the pharmacological action of the proteins in the vertebrate host and to test their potential for drug development.
- To uncover the structural basis of the stringent target specificity of arthropod salivary protease inhibitors; arthropod protease inhibitors are active against a limited number of vertebrate serine and cysteine proteases.
Ixodes scapularis salivary cystatins facilitate tick blood feeding and pathogen transmission
Two cystatins that are secreted from Ixodes scapularis salivary glands were characterised. The first cystatin was named sialostatin L, because it displays inhibitory activity against cathepsin L in vitro. Using an in vivo animal model for acute inflammation, combined with cell culture and biochemical assays, we attributed an anti-inflammatory and immunosuppressive action of this protein to the vertebrate host. Subsequently, the characterization of a second salivary cystatin followed, which was named sialostatin L2 to emphasize its redundant inhibitory activity against cathepsin L. Both cystatins are encoded by two loci in the tick genome, they are not equally potent in the inhibition of some papain-like enzymes and they also differ in antigenicity. Sialostatins L and L2 are the first cystatins described to date that display a highly specific affinity for a relatively limited number of cathepsins and having produced their crystals, we demonstrated the structural basis for their stringent specificity. Given the absence of classical genetic approaches to this non-model arthropod vector, we undertook a reverse genetic approach to silence sialostatins in ticks using RNAi techniques. The reduction of cystatin transcripts in I. scapularis salivary glands resulted in tick feeding inhibition, reduced tick size/weight and reduced number of eggs laid. Moreover, when normal ticks were exposed to a rabbit previously infested with sialostatin silenced ticks, they exhibited significant feeding impairment due to an enhanced host immune response, verifying the immunomodulatory properties of the proteins in vivo. Due to the observed tick feeding impairment in the gene silencing experiments, animals were vaccinated with sialostatin L2 to test whether a similar protective effect against tick infestation can be achieved. We worked with tick nymphs because their small size makes it difficult to notice their attachment to a host and to remove them before pathogen transmission takes place. Three times more nymphs failed to attach or blood feed on the sialostatin L2 vaccinated group. The rest of the nymphs that managed to feed on vaccinated animals, received lesser blood in comparison to the control group, because an average tick repletion weight of 1.9 mg was achieved for the nymphs fed on sialostatin L2 vaccinated animals, compared to the corresponding 2.8 mg for the nymphs fed on the control group. These results revealed an underlying potential for sialostatin L2 as a protective antigen for the development of an anti-tick vaccine. Moreover, we demonstrated that sialostatin L2, but not sialostatin L, facilitates Borrelia transmission in mice. Next, we produced the recombinant cystatins in LPS (lipopolysacharide) free formulation to further investigate the mechanism(s) by which they affect vertebrate immunity. Sialostatin L inhibits OVA (ovalbumin)-specific CD4+ T cell proliferation. The production of IFN-gamma (interferon-gamma) was diminished by 50% in both OVA- and Con A (concanavalin A)-dependent proliferation assays. Additionally, LPS-induced TNF-alpha (tumour necrosis factor-alpha) and IL-12 (interleukin-12) production, as well as CD80 and CD86 expression by dendritic cells were inhibited by sialostatin L, suggesting an action of the protein on antigen-presenting cells that ultimately leads to T cell proliferation inhibition. Finally it was demonstrated that sialostatin L exerts its action through the inhibition of vertebrate cathepsin S activity, by employing different in vivo animal models for antigen induced T cell proliferation and by using cathepsin knock out mice. Actually it was further demonstrated in an animal model of multiple sclerosis that sialostatin L can have a preventive role in disease onset.
Transcriptomic approaches in arthropod salivary glands
Our expertise in the field was gained at the Laboratory of Malaria and Vector Research (LMVR) at the National Institutes of Health in the USA and under the mentorship of Professors Ribeiro and Valenzuela. Michail Kotsyfakis, Jindrich Chmelar and Alexandra Schwarz are now specialized in the experimental procedures to study the salivary gland transcriptome from various arthropods. More specifically we are familiar with tissue isolation, total RNA and mRNA extraction, cDNA production and library construction. This work resulted in our co-authorship in four already published sialotranscriptome projects addressing the salivary transcriptome of the ticks Ixodes ricinus and Ornithodoros coriaceus, the triatomine Triatoma infestans and the tree hole mosquito Ochlerotatus triseriatus.
Salivary antigens of Triatoma infestans
Chagas disease (American trypanosomiasis) is one of the six most important tropical diseases in the world and is endemic in Latin America. Vector control programmes using insecticides against the most effective vector, Triatoma infestans were highly successful in significantly reducing domestic triatomine populations. Nevertheless, Chagas disease is not fully controlled yet and re-emergence of triatomines is a continuous threat in many areas of Latin America. Therefore, new methodologies are required to detect re-emerging T. infestans populations at an early stage and for sustained, long-term monitoring of previously endemic Chagas disease regions. Salivary proteins elicit an antibody response in their hosts and this can be used to measure exposure of domestic and peridomestic hosts to triatomines. Hence, anti-saliva antibody responses of chickens and guinea pigs to salivary proteins of a small number of T. infestans were characterised and highly immunogenic antigens were identified. rTiSP14.6, a 14.6 kDa salivary protein of T. infestans [gi|149689094] was synthesised as recombinant protein and evaluated successfully to be a triatomine specific and promising epidemiological marker for the detection of low numbers of different triatomine species in peridomestic habitats using chicken sera. This marker may be a useful tool in Chagas disease surveillance.
List of people of Laboratory: Kotsyfakis Lab
List of publications of Laboratory: Kotsyfakis Lab