The Roles of Two Clip Domain Serine Proteases in Innate Immune Responses of the Malaria Vector Anopheles gambiae

Jennifer Volz,Mike A Osta,Fotis C Kafatos,Hans-Michael Müller

doi:10.1074/jbc.m506191200

Jennifer Volz, Mike A Osta + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m506191200

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Abstract

The malaria vector Anopheles gambiae is capable of multiple immune responses against Plasmodium ookinetes. Accumulating evidence in several insect species suggests the involvement of serine protease cascades in the initiation and coordination of immune responses. We report molecular and reverse genetic characterization of two mosquito clip domain serine proteases, CLIPB14 and CLIPB15, which share structural similarity to proteases involved in prophenoloxidase activation in other insects. Both CLIPs are expressed in mosquito hemocytes and are transcriptionally induced by bacterial and Plasmodium challenges. Functional studies applying RNA interference revealed that both CLIPs are involved in the killing of Plasmodium ookinetes in Anopheles. Studies on parasite melanization demonstrated an additional role for CLIPB14 in the prophenoloxidase cascade. We further report that both CLIPs participate in defense toward Gram-negative bacteria. Our findings strongly suggest that clip domain serine proteases serve multiple functions and play distinctive roles in several immune pathways of A. gambiae.

Highlights

Melanization is a potent insect defense response, in which activation of a serine protease cascade leads to proteolytic conversion of prophenoloxidase (PPO) into active phenoloxidase (PO)
Cloning and Molecular Characterization of CLIPB14 and CLIPB15— To obtain full-length sequences of CLIPB14 and CLIPB15, a cDNA library (4a-2/3B) constructed from an A. gambiae cell line was screened with CLIPB14- and CLIPB15-specific probes that were amplified from the same cDNA library, using a gene-specific reverse primer and a T3 primer
CLIPB14 and CLIPB15 belong to subfamily B clip domain serine proteases, which are most similar in sequence architecture to the known PPO-activating CLIPs [5,6,7, 9] of Holotrichia diomphalia, Bombyx mori, and Manduca sexta (Fig. 1A)

Summary

Introduction

Melanization is a potent insect defense response, in which activation of a serine protease cascade leads to proteolytic conversion of prophenoloxidase (PPO) into active phenoloxidase (PO). In certain genetic backgrounds of Anopheles gambiae (the major vector of human malaria in sub-Saharan Africa), notably the L3-5 strain, activation of the PPO cascade leads to melanotic encapsulation of Plasmodium ookinetes as they egress from the midgut epithelium; this phenotype is a prime example of refractory mechanisms associated with a compromised parasite cycle [11,12,13]. In susceptible strains such as G3, parasite melanization is absent or extremely rare. Our findings suggest that individual CLIPs can serve multiple reactions, suggesting the existence of cross-talk between distinct mosquito immune responses

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