Functional and Molecular Characterization of Anopheles gambiae IMD/Rel2 Pathway in Anti-Microbial Defense

Abstract

Mosquitoes transmit several pathogens of global public health concern. Anopheles mosquitoes are vectors for malaria, one of the deadliest diseases worldwide. Like all other organisms, Anopheles mosquitoes are equipped with a highly efficient innate immune system to defend against the potentially harmful microorganisms to which they are exposed in their natural habitats. The expression of antimicrobial peptides (AMPs) by barrier epithelia and immune cells is an evolutionary conserved innate immune response of metazoans. Studies in model dipterans identified two key immune signaling pathways that protect against systemic infections: the Toll and the immune deficiency (IMD) pathways. These pathways regulate the expression of numerous immunity and non- immunity genes by activating NF-κB transcription factors. In the Afrotropical malaria vector Anopheles gambiae s.l., the IMD pathway plays pleiotropic roles in immunity, including resistance to malaria parasites, which are mediated by its NF-kB transcription factor Rel2. Rel2 exists as a full-length form (Rel2-F) containing the Rel-homology domain (RHD) and the C-terminal inhibitory ankyrin (ANK) and death domains (DD), and a shorter alternatively spliced form (Rel2-S) proposed to encode a constitutively active protein containing only the RHD. Despite its important roles in immunity, there are still multiple uncertainties concerning the identity and function of key components of the IMD pathway, as well as its overall contribution to mosquito resistance to systemic bacterial infections. Here, using in vivo reverse genetic analysis by RNA interference (RNAi), we show that Rel2 is critical for limiting the burden of Gram-negative and Gram- positive bacterial proliferation in An. gambiae G3 strain after systemic infections and this function is attributed to the endoproteolytic activation of Rel2-F in the fat body but not to Rel2-S. Interestingly, while Rel2-F activation in the fat body regulates the expression of Cecropin 1 and Defensin 1, its activation in the midgut after oral infections is dispensable for their regulation. We provide direct evidence that PGRPLC1 is necessary and sufficient for Rel2-F activation in the fat body in response to infections with Gram- positive bacteria containing Lysine-type peptidoglycan, however sensing of Gram- negative bacteria and Gram-positive bacilli containing DAP-type peptidoglycan is more complex and may be mediated by various PGRPLC isoforms. We also show that, in the mosquito fat body, Rel2-F activation cleavage is dependent on the adaptor protein Immune Deficiency (Imd) and the IkB Kinase (IKK) complex but seems to be independent of several other canonical intracellular pathway components. Altogether, our data demonstrate that the mosquito IMD pathway incorporates distinct receptor modules to detect both Gram-positive and Gram-negative bacterial infections and may adopt a non-canonical intracellular signaling cascade that regulates Rel2-F activation cleavage.