Abstract:
Human malaria is the most important disease in tropical countries in terms of morbidity and mortality. Malaria transmission involves complex interactions between Plasmodium falciparum and Anopheles gambiae. For successful establishment of invasion/infection of the Anopheles gambiae midgut the parasite must overcome the immune responses of the vector. Matrix metalloproteinase (MMPs) are a family of zinc metalloendopeptidases known to disrupt sub-endothelial membranes, destroy tight junctions and shed active cytokines, chemokines and other MMPs through cleavage from their precursors. The latter function putatively explains the great parasite loss during invasion of the Anopheles gambiae midgut by the parasite. The objective of this thesis was to study matrix metalloproteinases in An. gambiae as a potential addition to transmission blocking strategies. BLASTp searches of the complete Anopheles gambiae genome using Drosophila melanogaster MMP resulted in the identification of two Metazoa-like MMP genes. Domains of these proteases were determined through InterProScan. The 3-D structure was determined using MODELLER. The structure was validated using MetaMQAPII, ProSA and PROCHECK. A validation of the presence of MMPs in Anopheles gambiae was performed through RNA extraction, cDNA synthesis and Polymerase Chain Reaction amplification. Based on the BLAST output, two MMP genes similar to Drosophila melanogaster MMP were found in An. gambiae (AGAP006904 and AGAP003929). The proteases were shown to have a prodomain, metalloproteinase domain (catalytic domain) and a hemopexin domain and were classified into superfamily and family through presence of conserved domains and residues in a multiple sequence alignment (MSA). The modeled protein had a similar structural conformation to human pro-collagenase. The results of the amplification showed that AGAP006904 produced a truncated transcript. PCR amplification showed that MMP1 transcript A (AGAP006904) is expressed in the larvae, pupae and adult of An. gambiae. We can conclude that, Anopheles MMP is similar to MMP from humans and Dipterans in structural conformation and domain architecture. The presence of MMP in the 3 stages of Anopheles gambiae indicates a possible role in development. Knowledge of the structure and activation of Anopheles MMP is vital in understanding how this protein folds, which is vital in coming up with transmission blocking strategies to either inhibit or activate these proteases.
