Effects of Malaria Infection in Midgut Phosphotyrosine Profile of Aedes aegypti

Many events are observed in mosquito females in response to a blood meal infected with the malaria parasite. Several signaling cascades are probably triggered by infection, most of which are still unknown. The final target of every cell-signaling pathway involves the reversible phosphorylation of proteins. Over the past two decades, it has become clear that tyrosine phosphorylation plays a pivotal role in a variety of important signaling pathways in multicellular organisms. In addittion malaria parasite genome lacks classical tyrosine kinases. Given the importance of tyrosine phosphorylation, the present study was designed to identify mosquito proteins whose tyrosine phosphorylation state is modified by malaria parasite. Aedes aegypti females were bood-fed in Plasmodium gallinaceum infected-chickens. Twenty hours after blood meal, the midguts were dissected and blotted against anti-phosphotyrosine. A decrease was observed on the total tyrosine phosphorylation in infected midguts. The same effect was observed when SDS-PAGE gels were treated with Pro Q Diamond, a fluorescent staining for phosphoproteins. Identification of such phosphoproteins is currently being conducted by 2D electrophoresis coupled to mass spectrometry. We also employed tyrosine phosphatase assays as an overall sensor of changes on tyrosine phosphorylation. A significant increase was observed in midgut tyrosine phosphatase activity 20 hours after infected blood meal when compared with control. A tyrosine phosphatase gene sequence from A. aegypti midgut was used to design a primer and follow gene expression in unfed female midgut.  Analysis of gene expression will be conducted in both blood and infected blood fed midguts by RT-PCR. Detailed knowledge of these signaling mechanisms may be exploitable in the future towards developing novel strategies of blocking malaria transmission. Supported by CNPq, OMS, FAPERJ and IFS.