Functional and molecular characterization of CLIPA7 in Anopheles gambiae immunity.

Abstract

Malaria is one of the most devastating vector-borne diseases in the world. It is caused by parasites of the genus Plasmodium that are transmitted to humans by the bite of female Anopheles mosquitoes. Studies in the major African vector A. gambiae revealed that mosquitoes are not passive to Plasmodium infection, rather they mount potent immune responses which are particularly efficient against ookinete stages of the parasite. Clip domain serine proteases and their homologs are important components of mosquito immune responses and have been particularly studied in the context of melanization and complement regulation. In this project, we characterized the role of the clip domain serine protease homolog CLIPA7 which was previously identified as a negative regulator of the melanization response to P. berghei ookinetes. CLIPA7 has an unusual protein structure that contains, in addition to the clip domain, several other protein-protein interaction domains including a proline rich domain, a tropoelastin and prion-like domains which indicate that it might play a broader role in mosquito immunity compared to other homologs. We revisited the role of CLIPA7 in defense against P. berghei infections and showed that its knockdown result in a more pronounced melanization response against ookinetes than previously reported. This response is dependent on the complement-like protein TEP1 protein which is a key player in mosquito immunity. Our data also revealed that CLIPA7 is not involved in resistance to S. aureus and E. coli infections, however, CLIPA7 Kd mosquitoes exhibited drastically enhanced tolerance to S. aureus but not to E. coli infections. Biochemical analysis revealed that CLIPA7 regulates the activation cleavage of both CLIPA8 and CLIPA14, which are key positive and negative regulators of the melanization response, respectively. This suggests that CLIPA7 plays a complex role in the regulation of the clip cascades. Genetic and biochemical analysis of clip domain serine proteases and their homologs is needed to build