Malaria continues to expand as a major public health threat throughout the world. The spread of drug resistant malaria and the lack of an effective vaccine make alternative and complementary approaches to malaria control increasingly important. The overall theme of this research program is to develop methods of blocking malaria transmission by interfering with specific Plasmodium-mosquito molecular interactions within the mosquito midgut. The focus of this project is to determine the mechanisms by which Plasmodium chitinase(s) allow the ookinete to penetrate the chitin- containing peritrophic matrix, thereby initiating mosquito midgut invasion. The underlying hypothesis is that interfering with the function of Plasmodium chitinase(s) can prevent malaria transmission. The first specific aim is to develop methods of blocking malaria transmission to mosquitoes by interfering with Plasmodium chitinases. Both vaccine and rational drug discovery approaches will be pursued. The second specific aim is to use targeted disruption of the recently identified P. falciparum chitinase gene, PfCHT1, to assess the role of this chitinase gene product in mosquito peritrophic matrix penetration. Constructs will aim at interrupting the catalytic domain of the PfCHT1 gene. The phenotype of chitinase-knockout parasites will be assessed by oocyst enumeration and with immunofluorescence and electron microscopy. The third specific aim is to study the cell biology of the chitinase within the ookinete. Immunofluorescence and immunoelectron microscopy will be used to identify the ookinete organelles with which the chitinase is associated. Identification of the mechanisms of intracellular trafficking and proteolytic post-translational modifications may allow for the development of additional molecular targets of interfering with chitinase function. These studies will further elucidate the mechanisms by which chitinase allows the ookinete to initiate invasion of the mosquito midgut. More practically, these studies are expected to determine whether Plasmodium chitinases will be useful targets of blocking malaria transmission.