Final Activity Report Summary - A. GAMBIAE C-LECTINS (The role of C-type lectins in A. gambiae immunity and vector-Plasmodium interactions)
Malaria remains an important health problem responsible for the death of up to 3 million people annually, largely children between the age of one and five in sub-Saharan Africa. The disease is transmitted from infectious female Anopheles mosquitoes to humans through the sporozoite stage of the protozoan parasite, Plasmodium. During its journey in the mosquito, Plasmodium suffers severe losses in parasite numbers due to immune defence mechanisms mounted by the vector. However, evidence for the direct implication of mosquito immune genes in parasite development was only recently provided and that due to the establishment of RNAi-mediated gene silencing technique in the major malaria vector in Africa, A. gambiae.
Using RNAi we have first identified two C-type lectins, CTL4 and CTLMA2, which act as agonists of parasite development, protecting the parasite from the potent immune response of the vector, melanisation. The knockdown of either lectin, in particular CTL4, induced the massive melanisation of P. berghei ookintes in the subepithelial space of the mosquito midgut. These lectins are secreted into the mosquito hemolymph in the form of a disulfide-linked heterodimer. Melanisation requires the proteolytic conversion of inactive prophenoloxidase (PPO) into active phenoloxidase (PO), a key enzyme in melanin formation. Hence, the lectins are most probably involved in blocking (either directly or indirectly) PPO activation. In the ultimate aim of unravelling the mechanism of action of CTL4 and CTLMA2 we took a candidate approach to identify the proteins that may potentially interact with these lectins. By analogy to published data from other invertebrates, in particular the silkworm Manduca sexta, we hypothesised that CTL4 and CTLMA2 might associate with Masquerade-like, non-catalytic, clip-domain serine proteases (known also as serine protease homologues, SPH) of the A. gambiae CLIPA subfamily in order to block the vector melanisation response. SPHs characterised from other insects species such as M. sexta and Holotrichia diomphalia were shown to act as cofactors for Easter-like, catalytic clip-domain serine proteases that ultimately catalyse the limited proteolytic cleavage of PPO to PO. We utilised a systematic in vivo RNAi-mediated reverse genetic screen of 10 CLIPA (Masquerade-like) and 11 CLIPB (Easter-like) genes of A. gambiae in order to identify the gene module that regulates (either promote or dampen) ookinete melanisation in the mosquito midgut and to identify potential interacting partners with CTL4 and CTLMA2.
Our data revealed that several but not all CLIPB genes promote Plasmodium melanisation, exhibiting partial functional overlap and synergy. We also reported that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanisation, while three other CLIPAs (CLIPA2, CLIPA5 and CLIPA7) are novel synergistic inhibitors of this response.
The simultaneous knockdowns of CLIPA2 and CLIPA5 induced massive ookinete melanisation, a similar phenotype to that of the CTL knockdowns, suggesting that CTL4 and CTLMA2 may interact with these inhibitory SPHs forming a complex that blocks PPO activation. Importantly, the roles of certain CLIPAs and CLIPBs are strain-specific, indicating that the composition of the melanisation module differs between strains. We also provide evidence that in susceptible mosquitos' melanisation induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.
Using RNAi we have first identified two C-type lectins, CTL4 and CTLMA2, which act as agonists of parasite development, protecting the parasite from the potent immune response of the vector, melanisation. The knockdown of either lectin, in particular CTL4, induced the massive melanisation of P. berghei ookintes in the subepithelial space of the mosquito midgut. These lectins are secreted into the mosquito hemolymph in the form of a disulfide-linked heterodimer. Melanisation requires the proteolytic conversion of inactive prophenoloxidase (PPO) into active phenoloxidase (PO), a key enzyme in melanin formation. Hence, the lectins are most probably involved in blocking (either directly or indirectly) PPO activation. In the ultimate aim of unravelling the mechanism of action of CTL4 and CTLMA2 we took a candidate approach to identify the proteins that may potentially interact with these lectins. By analogy to published data from other invertebrates, in particular the silkworm Manduca sexta, we hypothesised that CTL4 and CTLMA2 might associate with Masquerade-like, non-catalytic, clip-domain serine proteases (known also as serine protease homologues, SPH) of the A. gambiae CLIPA subfamily in order to block the vector melanisation response. SPHs characterised from other insects species such as M. sexta and Holotrichia diomphalia were shown to act as cofactors for Easter-like, catalytic clip-domain serine proteases that ultimately catalyse the limited proteolytic cleavage of PPO to PO. We utilised a systematic in vivo RNAi-mediated reverse genetic screen of 10 CLIPA (Masquerade-like) and 11 CLIPB (Easter-like) genes of A. gambiae in order to identify the gene module that regulates (either promote or dampen) ookinete melanisation in the mosquito midgut and to identify potential interacting partners with CTL4 and CTLMA2.
Our data revealed that several but not all CLIPB genes promote Plasmodium melanisation, exhibiting partial functional overlap and synergy. We also reported that several CLIPA genes have contrasting roles: CLIPA8 is essential for parasite melanisation, while three other CLIPAs (CLIPA2, CLIPA5 and CLIPA7) are novel synergistic inhibitors of this response.
The simultaneous knockdowns of CLIPA2 and CLIPA5 induced massive ookinete melanisation, a similar phenotype to that of the CTL knockdowns, suggesting that CTL4 and CTLMA2 may interact with these inhibitory SPHs forming a complex that blocks PPO activation. Importantly, the roles of certain CLIPAs and CLIPBs are strain-specific, indicating that the composition of the melanisation module differs between strains. We also provide evidence that in susceptible mosquitos' melanisation induced by knockdown of either CTL4 or CLIPA2/CLIPA5 directly kills ookinetes, in contrast to refractory mosquitoes where it merely disposes of dead parasites.