The aim of this research program is to investigate the anti-hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easy site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones is found by expression in bacterial or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced, providing new insights in the discovery of novel compounds. ? ? In the current fiscal year we have advanced our knowledge on novel proteins in saliva of blood feeding ticks and mosquitoes. Specifically, we discovered the bioamine binding properties of an unique family of proteins only found in the saliva of blood-sucking flies (1), characterized the target specificity of a salivary cysteine protease inhibitor from the tick vector of Lyme disease in the Eastern US, which also has anti-inflammatory properties (2), characterized an inositol phosphatase from the saliva of kissing bugs that has unique kinetic properties (3), discovered a new salivary enzymatic activity (never reported before, a salivary endonuclease) in the common house mosquito vector of West Nile fever (4), and further characterized the action of Ixolaris, a tick salivary anti-clotting that we have previously discovered (5). A propos, Ixolaris was patented last July 18th (14).? ? We have also increased our salivary transcriptomes database by expanding the coverage of EST?s in the salivary transcriptomes of the main malaria vector in Africa, Anopheles gambiae, and the tick vector of Lyme disease, Ixodes scapularis (6-8). We have also described the salivary transcriptomes of adult male An. gambiae (7). Because male mosquitoes do not blood feed, the subtraction of the male transcriptomes from the female transcriptomes allow identification of transcripts that have high probability of acting on the blood meal function of the salivary gland. These databases allow us to map our ignorance regarding the pharmacological potential of hematophagous arthropods: There are over 70 secreted salivary proteins in mosquitoes, and over 200 in ticks, but we know the function of less than 15% of these proteins. Additionally, many protein families are completely novel, no similarities being found to any other known proteins. These transcriptomes are now the substrate for many years of research to be followed by many laboratories.? ? Our bioinformatic capability allowed us to collaborate extensively with other research groups inside and outside the NIAID. Accordingly, we collaborated with Dr. Anthony James (UC-Irvine) for the development of a new tool to identify promoter sequences in An. gambiae (9), and to annotate gene expression profiles in the same mosquito (11); we helped Dr. Vernick?s laboratory to annotate a new proteome set of An. gambiae based on a new gene prediction algorithm (10), and contributed to the sequencing and annotation of transcriptomes of mosquito and cockroach corpora allata, an important organ involved in hormonal control of molt and egg development in insects (12).? ? Finally, we have also collaborated with field work being done in Mali by the NIAID intramural team, where the PI helped to adapt micro techniques for protein measurement in just-hatched mosquito larvae (13).
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