Abstract
BackgroundClip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies—CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE—based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22.MethodsClustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated.ResultsWe performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria.ConclusionsOur findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity.Graphical
Highlights
Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects
Multiple amino acid sequence alignment Multiple amino acid sequence alignment showed that Ae. aegypti CLIPB15 is similar to An. gambiae CLIPB15, Bombyx mori proPO activating enzyme (PPAE) and Drosophila melanogaster GRASS; and that Ae-CLIPB22 is similar to Bombyx mori PPAE and An. gambiae CLIPB15 (Fig. 1)
The transcript expression levels of AeCLIPB22 had significantly increased by the fourth-instar larval stage compared to the first-instar [first-instar vs fourth-instar, t-test, t(22) = 17.67, P < 0.0001] and the higher level was maintained in the pupal and adult female mosquito stages
Summary
Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immunity plays an important role in limiting pathogen infection through the production of molecules such as antimicrobial peptides (AMPs), through phagocytosis and encapsulation, and by the secretion of physical barriers and melanization [5]. Melanization is an immune response of arthropods to a wide range of viruses [6], bacteria [7, 8], fungi [9], nematodes associated with chromogenic bacteria [10] and other eukaryotic parasites [11,12,13] This immune response is regulated by prophenoloxidase (proPO), which mediates the conversion of tyrosine to melanin; recognition of a pathogen triggers a serine protease cascade in which activated serine proteases cleave proPO to produce phenoloxidase (PO) [14]. In Ae. aegypti, the activation of genes coding for AMPs and other immune effectors is achieved by releasing nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2, which are homologous to those in Drosophila melanogaster [16]
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