Mosquito Midgut Immune Responses &Malaria Transmission

Niaid Extramural Activities

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Abstract

We are interested in exploring the interactions between the mosquito (A. gambiae and A. stephensi) immune system and the malaria parasite and to understand how they determine vector competence. Some major areas of interest include: Cell biology of ookinete invasion. Detailed studies of the cellular responses of mosquito midgut epithelial cells to parasite invasion in the An. stephensi-P.berghei system revealed that parasites inflict extensive damage as they migrate through midgut epithelial cells, which ultimately leads to genome fragmentation, nuclear picnosis and cell death. This studies led us to propose the Time-Bomb Model of Invasion, which states that ookinetes have a limited time window to escape unharmed from the invaded cell, as the cascade of responses mediating epithelial cell death are also potentially lethal to the parasite. Recent studies indicate that protein nitration is a two-step process involving NOS and inducible peroxidases. A publications describing this work has been submitted. We are currently raising antibodies against these enzymes using DNA vaccines and recombinant proteins to establish their subcellular localization and silencing their expression to determine how this would affect parasite survival. Comparative studies using the A. aegypti-P.gallinaceum model system revealed that the parasite-induced damage to epithelial cells during invasion is repaired by a different repair mechanism and does not involve NOS or peroxidase induction. The role of Reactive Oxygen Species (ROS) in An. gambiae refractoriness to malaria infection. A genetically selected refractory strain of A. gambiae blocks Plasmodium development, melanizing and encapsulating the parasite. Morphological, microarray mRNA and physiological studies indicate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady state levels of ROS, which favor melanization of parasites as well as Sephadex beads. Dietary supplementation with antioxidants reduced parasite and bead encapsulation. We are currently silencing catalase expression to determine if decrease clearance of ROS decreases parasite survival. The role of the STAT pathway in epithelial responses to malaria infection. Band shift experiments from midgut nuclear extracts indicate that parasite invasion activates the STAT (Signal Transducers and Activators of Transcription) pathway. We have characterized two members of this family of transcription factors in A. gambaie as well as to repressors, SOCS and PIAS, which are induced by malaria infection at the transcriptional and translational level, respectively We are currently performing a series of dsRNA silencing experiments in vivo and in tissue culture to establish the relevance of the STAT pathway in the outcome of parasite midgut infection. Trypsin activity and dengue-2 (DEN-2) midgut infection in Aedes aegypti. The role of midgut trypsin activity on viral invasion and replication was investigating by administering soybean trypsin activity in vivo. Trypsin was found to play a critical role regulating viral replication at the level of transcription and translation. Direct contact of trypsin with the virus also transiently enhanced the association of DEN-2 with the midgut epithelium, suggesting that proteolytic processing of the viral surface increases infectivity.