Vector arthropods, such as mosquitoes, triatomine bugs and ticks, salivate while they puncture our skin in their search of blood. This saliva contains dozens to hundreds of compounds that have anti-clotting, anti-platelet, vasodilatory, anti-inflammatory, and immunomodullatory functions. While helping the vector to feed, it also modifies the site where pathogens are injected and in many cases facilitates the infection process. For this reason, salivary proteins of vectors can be used as vaccine targets for the diseases they transmit. Salivary proteins can also be used as immuno-epidemiological markers of vector exposure, and in themselves can have potent and novel pharmacological activities. Because the saliva of hematophagous animals is under attack of their host's immune system, their constituents are under a rapid evolutionary pressure in an arms'race scenario with their hosts, causing an enormous variety of unique protein families even in closely related organisms. The section of vector biology aims at uncovering the biodiversity of salivary proteins in the near 500 genera of blood sucking arthropods, and to discover the function of the novel protein families that we encounter. Accordingly we have developed a two pronged approach focusing in sialotranscriptome discovery projects and functional sialomic studies. In addition to these core studies on the saliva of vector arthropods, the section also collaborated with other members of the LMVR and other extramural scientists lending its expertise in bioinformatics, structural biology and vascular biology. In the current fiscal year (2014), members of the Section of Vector Biology contributed to a total of 22 papers and 2 patent applications. Sialotranscriptome discovery and evolution projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 1,000 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genera studied so far has several unique protein families. Indeed there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what is the universe of salivary proteins associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than 20 genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding, and identify proteins with pharmacological and vaccine potential. In the current fiscal years, we produced fiver papers related to sialotranscriptome and proteome discovery and evolution of hematophagous arthropods, including: for the flies Corethrella appendiculata (1) and Psorophora albipes (2), the first from these two genera, helped to understand the evolution of salivary protein-coding genes in flies, and from the ticks Rhipicephalus microplus (3) and the European Lyme disease vector, Ixodes ricinus (4-5). We have also shown that salivary protein coding genes from the malaria vector An. gambiae are under strong positive selection, supporting our hypothesis of accelerated evolution of these genes possibly due to the relentless immune response of their hosts. Functional studies: We advanced our knowledge regarding the function of vector salivary proteins as reported in 8 publications, describing a novel family of insect contact-pathway blood clotting inhibitors (7), two inhibitors of platelet aggregation acting on the collagen pathway (8-9), and showing that this family of proteins is at least 200 million years old in blood feeding flies, a novel salivary endonuclease from sand flies that affect blood feeding and Leishmania transmission (10), a novel factor XIa inhibitor from bat saliva (11), a tick salivary serine protease that activates protein C (12), and characterized the binding and enzymatic properties of a bilin-binding protein from a venomous caterpillar (13). The anti-Plasmodial properties of the anti-oxidant tempol was also studied (14). Bioinformatic collaborations: Dr. Ribeiro collaborated with intra and extra mural investigators lending his expertise in bioinformatics, helping to functionally characterize the sialotranscriptome changes of a tick following knockdown of the selenocysteine-specific elongation factor and its effect on Rickettsia parkeri burden (15), novel proteins in the milk glands of tsetse vectors of African trypanosomiasis (16), seminal proteins in the Asian tiger mosquito Aedes albopictus (17), the transcriptome and developmental regulation of gene expression in the malaria vector An. aquasalis (18), the in-depth annotation of midgut transcriptomes of the malaria and Chagas disease vectors An. gambiae (19) and Rhodnius prolixus (20), the mevalonate pathway in the sex pheromone glands of a sand fly vector of Leishmaniasis (21), and the discovery of a novel flavivirus infecting cattle in Brazil (22). Patent application: There were two patent applications filed this fiscal year, one for an unique inhibitor of factor XIa from bats (23) and another submitted in Brazil regarding anti-tick vaccines for cattle (24). During this fiscal year, members of Dr. Ribeiro laboratory were awarded four patents from the US Patent and Trademark Office as follows: 1. Fischer, L.B., Valenzuela, J.G., Ribeiro, J., Kamhawi, S. (submitted May 6, 2009) Leishmania vaccine using sand fly salivary immunogen. US Department of Commerce, Patent and Trademark Office. Patent number 8,603,808 issued on December 10, 2013. 2. Valenzuela, J.G., Ribeiro, J.M.C., Barral, A., Netto, M., Brodskyn, C., Gomes, R. (submitted January 7, 2009) Lutzomyia longipalpis polypeptides and methods of use. US Department of Commerce, Patent and Trademark Office. Patent number 8,628,780 issued on January 14, 2014. 3. Valenzuela, J.G., Belkaid, Y., Kamhawi, S., Sacks, D., Ribeiro, J.M.C. (submitted December 11, 2012) Anti-arthropod vector vaccines, methods of selecting and uses thereof. US Department of Commerce, Patent and Trademark Office. Patent number 8,629,260 issued on January 14, 2014. 4. Francischetti, I.M.B., Monteiro, R.Q., Ribeiro, J.M. (submitted March 18, 2010) Use of ixolaris, a tissue factor inhibitor, for the treatment of cancer. US Department of Commerce, Patent and Trademark Office. Patent number 8,772,238 issued on July 8, 2014.
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