Functional Characterization of Anopheles gambiae Spätzle Gene Family

Abstract

Mosquito vectors of diseases are not passive hosts for the pathogens they transmit, rather they employ a robust innate cellular and humoral immune responses against the various microbes they encounter. In Drosophila and other insect species, a key immune signaling pathway, the Toll pathway, provides resistance against fungal and bacterial infections, mainly Gram-positive bacteria, and is a major regulator of the expression of several immunity genes, specifically those encoding antimicrobial peptides (AMPs), as well as other genes involved in the stress response. In insects, Toll is activated by binding to a cleaved active form of the cytokine molecule Spätzle (Spz). Spz cleavage is regulated by a cascade of clip-domain serine proteases (CLIPs). Despite being well characterized in Drosophila, our knowledge of the Toll pathway activation remains largely fragmented in the major African malaria vector Anopheles gambiae, and mosquitoes in general, specifically that several components of this pathway have not been characterized yet.

Here, we functionally characterize by RNA interference (RNAi) the roles of the six Spz genes (Spz1-6) identified in the A. gambiae genome in immune responses to Grampositive and Gram-negative bacterial and fungal systemic infections. In the context of mosquito tolerance to Staphylococcus aureus infections, silencing of Spz genes resulted in mixed phenotypes, with Spz2 and Spz4 knockdown (kd) compromising survival in more trials compared to other candidates. Similarly, Spz2 kd significantly compromised mosquito survival to fungal infections in most of the conducted trials, while the other Spz genes gave variable phenotypes, suggesting that Spz2 may be indispensable for mosquito tolerance to fungal infections, in addition to its less prominent role against Gram-positive bacterial infections. However, none of the Spz gene kd compromised mosquito survival to Serratia marcescens systemic infections, suggesting that the Toll pathway may not be a key player in the immune defense against Gram-negative bacteria in A. gambiae. Spz gene silencing did not alter mosquito resistance to bacterial and fungal infections, indicating that the Toll pathway may not play a major role in bacterial and fungal clearance in the mosquito, as it does in Drosophila. QRT-PCR analysis revealed that most of the Spz genes were upregulated after fungal infections, and particularly peaked at 48 hours after infection, whereas none were induced after S. aureus challenge, revealing that the Toll pathway may be more implicated in antifungal defenses. A better understanding of the activation of the mosquito Toll pathway, its relevance to host defense, and the functional characterization of its components contributes significantly to our knowledge on mosquito-pathogen interactions, which would be especially relevant in the context of fungal infections since entomopathogenic fungi are being considered as potential biopesticides for the control of mosquito vectors of disease. Hence, a better characterization of this pathway would help understand the potential mechanisms that may be used by fungi to evade mosquito immune responses.