The Vector Molecular Biology Unit focuses on the molecular aspects of arthropod vector salivary and midgut proteins in vector/host and vector/parasite interactions. The two main hypotheses driving the research of this unit are:? ? 1) Cellular immune responses to vector arthropod salivary proteins produce an inhospitable environment in the skin of the host to the co injected pathogen, resulting in indirect killing or unsuccessful establishment in the vertebrate host. Identifying the vector salivary proteins and the correlates of protection particularly the initial immunological events will help us to understand the immunologic basis of protection and to select vaccine candidates to prevent pathogen transmission.? 2) Specific molecular interactions between the sand fly midgut and the Leishmania parasite are required for Leishmania survival and development to the infective stage in the insect vector. Characterization of these molecular interactions will help in the understanding of the molecular basis of Leishmania sand fly interactions and may identify a suitable target for a transmission blocking vaccine. ? ? The three main objectives of the unit are: ? ? I. Development of functional genomic tools to study immune responses to vector salivary proteins and their role in pathogen transmission.? II.Development of transcriptome and functional genomics tools to study the molecular interactions between sand fly midgut proteins and the Leishmania parasite.? III.Epidemiologic studies of leishmaniasis in Mali directed toward the understanding of the role of immune responses to vector saliva and Leishmania infection in humans. ? ? The unit has successfully developed a comprehensive approach to isolate and characterize salivary and midgut proteins from different sand flies. The Unit randomly sequenced transcripts from three distinct high quality full-length female Phlebotomus papatasi midgut-specific cDNA libraries from sugar-fed, blood-fed and Leishmania major-infected sandflies. Furthermore, we compared the transcript expression profiles from the three different cDNA libraries by customized bioinformatics analysis and validated these findings by semi-quantitative PCR and real-time PCR. Transcriptome analysis of 4010 cDNA clones resulted in the identification of the most abundant P. papatasi midgut-specific transcripts. The identified molecules included those with putative roles in digestion and peritrophic matrix formation, among others. Moreover, we identified sandfly midgut transcripts that are expressed only after a blood meal, such as microvilli associated-like protein (PpMVP1, PpMVP2 and PpMVP3), a peritrophin (PpPer1), trypsin 4 (PpTryp4) , chymotrypsin PpChym2, and two unknown proteins. Of interest, many of these overabundant transcripts such as PpChym2, PpMVP1, PpMVP2, PpPer1 and PpPer2 were of lower abundance when the sandfly was given a blood meal in the presence of L. major. ? The Unit also sequenced five cDNA libraries of the midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present during sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi. Comparative analysis of the different cDNA libraries resulted in the identification of transcripts differentially expressed by blood feeding and digestion as well as the midgut transcripts differentially expressed by the presence of Leishmania infantum chagasi. ? Furthermore, we have developed a functional genomic approach to identify vector salivary proteins that can produce antibody and or cellular immune responses. The approach is based in the selection of transcripts coding for secreted proteins from a sand fly salivary gland cDNA library, cloning of these molecules into a DNA plasmid, and intradermal immunization of these DNA plasmids in animals to test for production of immune responses. With this approach we identified vector salivary proteins capable to produce a delayed skin response (DSR), a surrogate of cellular immune response and prevent cutaneous leishmaniasis in mice. The unit has also increased the capabilities to mass colonize Phlebotomus duboscqi and Lutzomyia longipalpis sand flies in our insectary facilities. We have also succeeded in infecting sand flies with either L. major or L. chagasi and these sand flies are able to transmit the Leishmania parasite to rodents and produce disease. This represents the most natural model to produce the disease and will be used to test our vaccine candidates.? The unit recently began a project in Mali where cases of cutaneous leishmaniasis have been reported and confirmed by members of this unit. The rational of moving to a field site is to test our hypothesis that immune responses to sand fly salivary proteins affect the outcome of Leishmania infection. This translational research component of the unit is a multidisciplinary approach based on entomology, molecular biology, bioinformatics, immunology and clinical studies. Because little is known about the epidemiology of cutaneous leishmaniasis in this country and few clinical studies are limited to cases reported by the C.N.A.M. dermatology clinic in Bamako; we did not have any indication of the prevalence and distribution of the disease and the sand fly vector responsible for the transmission. We therefore began with epidemiological studies of leishmaniasis in two suspected foci by conducting a prevalence survey using a leishmanin skin test; identification of sand fly vector species, and animal reservoir with the ultimate goal to identify sand fly salivary proteins recognized by the endemic human population and to test whether these immune responses are related to protection against Leishmania infection. This is a relatively new project for our unit; however, preliminary information has been obtained in the last year. The district of Baraouli, located about 180 Km from Bamako, was chosen upon finding active cases of cutaneous leishmaniasis in villagers during preliminary site visits. Two villages within the district of Baraouli were selected for this study: Kemena and Sougoula (3 miles apart from each other). Our results showed that the prevalence of leishmaniasis was 41% and the incidence was 9.5% when the two villages were combined. Entomologic studies on the two villages and other sites reveal the presence of a variety of sand flies from the genus Sergentomyia; these sand flies do not transmit Leishmania. We also found Phlebotomus dubosqci outside and inside houses where active leishmaniasis cases were present. Preliminary experiments have identified Phlebotomus papatasi infected with Leishmania major suggesting this sand fly is the vector on this foci. Furthermore we have trapped a series of rodents and preliminary information shows that the sera of Mastomys natalensis (African soft-furred rat) recognized proteins from the saliva of Phlebotomus duboscqi and also antigens from Leishmania major (isolated in Mali) suggesting this rodent as a potential Leishmania reservoir. Salivary glands from female Phlebotomus dubosqci caught in the field were dissected and stored. These salivary glands were transported to the laboratory at NIH, and mRNA was isolated and a cDNA library was prepared and sequenced. Bioinformatic analysis revealed the presence of the most abundant salivary proteins from this vector and potential antigens for our studies on this field site.
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