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, we produced 5 papers (1-5) describing the sialome of Triatoma infestans, a vector of Chagas disease (1), the flea vector of plague (2) of a non-blood feeding mosquito (3) and of soft ticks (4-5). The T. infestans sialome allowed the comparison of the expanded lipocalin family found in triatomines to that of two other triatomines with known sialomes, T. brasiliensis and Rhodnius prolixus, thus identifying the fast evolution of these proteins. Novel proteins were also discovered. The transcriptome of the flea Xenopsylla cheopis was the first flea transcriptome to be published, and it uncovered a large number of novel protein families. Because mosquitoes also feed on sugar meals, the sialotranscriptome of these insects contain products associated with both sugar and blood meal acquisition. The transcriptome of the non-blood feeding mosquito Toxorhynchites amboinensis allowed an insight into a transcriptome that is thus purely associated with sugar meals, and also with antimicrobial activity, because it is known that antimicrobials, such as lysozyme, are injected with saliva of non-blood feeding mosquitoes. Finally, soft tick transcriptomes allowed the discovery of many unique protein families, comparisons among known protein families, and an insight in the evolution of tick saliva.? ? Functional sialomic studies:? ? We advanced our knowledge regarding the function of several salivary proteins, as well as discovering novel salivary properties, as was the case of sand fly saliva inhibiting neutrophil migration (6), understanding the evolution of tick anti-clotting proteins (7), discovering a novel tick protein family that counteracts inflammatory cysteinyl-lukotrienes (8-9), characterization of a family of biogenic amine binding proteins from ticks (10), characterization of the crystal structure of a mosquito biogenic amine binding protein (11), and further use of the tick anticoagulant Ixolaris as a tool to understand human clotting (12) and adenovirus mediated gene transfer (13). ? ? Expertise capabilities spin off:? ? Dr. Francischetti expertise in hemostasis led this year to 2 review articles on malaria and coagulation (14-15), a follow up of an article authored by him in 2007 on tissue factor exposure by endothelial cells contacting Plasmodium falciparum infected erythrocytes. Our bioinformatic capability allowed us to collaborate with Dr. Warwick Britton, specifically to identify in the genome of Mycobacterium tuberculosis a family of cutinase proteins that has vaccine potential (16).
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