In our search for understanding the evolution of blood sucking by arthropods, we realize the methodology used has drastically changed in the past few years. Traditionally, the research process first identified a biological activity in saliva or salivary gland homogenates of a particular organism, and then proceeded to isolate that activity as a relatively pure entity to allow its molecular identification. In the case the activity derived from a protein, peptide fingerprinting allowed the design and use of nucleotide probes to clone the coding mRNA (in the form of a cDNA) and final identification of the peptide sequence;the clone also allowed the manufacture of recombinant protein for further studies. Nowadays, the process has reverted. cDNA libraries are constructed from salivary glands of blood sucking arthropods and mass sequenced. Bioinformatic analysis reveals the salivary transcriptome of these organisms, which contains many unique protein families with unknown properties. We then proceed to select clones for expression, bioassay screening and characterization. Accordingly, there are two processes used in our lab, first, the construction and analysis of salivary gland cDNA libraries, and second, the recombinant expression and characterization of these proteins. We have also been developing bioinformatic capabilities in the form of specific software to help direct our studies. Sialotranscriptome discovery 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 400 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 a dozen 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 year (2010), we produced a total of 21 peer reviewed papers and two review articles. Six of the papers describe sialomes, including the sialome of the tsetse fly (1), the first so far produced for this family of flies, of the brown dog tick (2), of the West Nile vector, Culex tarsalis (3), of the arbovirus vector Ochlerotatus triseriatus (4), of the common bed bug, Cimex lectularius (5), and of the south American black fly Simuliun nigrimanum (6). Several new protein families were discovered in the sialomes of the tsetse fly, black fly and tick, which awaits functional characterization. The black fly studied has been implicated with possibly triggering the autoimmune disease pemphigus foliaceus, and its sialome disclosure opens the possibility of testing the patients'immunoreactivity to defined fly antigens. Functional sialomic studies: We advanced our knowledge regarding the function of several salivary proteins, as well as discovering novel salivary properties. The molecular specificity of the mosquito anti-platelet protein Aegyptin has been studied and its use in thrombosis treatment investigated (7). A tick salivary protein which inhibits tissue factor activation of Factor X was shown to inhibit tumor growth and angiogenesis (8). A new class of antimicrobial peptides were discovered in ticks (10), and salivary proteins of a kissing bug vector of Chagas'disease were identified that can be used as epidemiological markers of triatomine infestation. We have also contributed to understand the feeding behavior of mosquitoes infected with Rickettsia-like Wolbachia organisms (9). Two review articles on platelet aggregation inhibitors of hematophagous animals and in the salivary composition of blood feeding insects were also written (22, 23). Expertise capabilities spin off: Our bioinformatic capability lead to collaboration with diverse studies, as follows: for helping annotating proteins from reproductive organs of the tsetse fly (12) and body louse (14), to identify salivary protein families of ticks (13, 16);to characterize transcription and gene annotation from anopheline mosquitoes vectors of malaria in Africa (15, 18), and to identify genes associated with nitric oxide metabolism in Plasmodium falciparum (17). Our expertise in several areas of physiology and pharmacology lead to collaborative studies and spin-off research (19-21).
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