Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

Abstract

Background Clip 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. Methods Clustal 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. Results We 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. Conclusions Our 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 Abstract

Genome resequencing and genome-wide polymorphisms in mosquito vectors Aedes aegypti and Aedes albopictus from south India

“…Furthermore, our data uncovers polymorphisms in genes of the CLIP B and D like serine protease subfamilies, corroborating earlier RNAseq reports [61]. Notably, CLIP-B is the major sub-family, members like CLIPB15, CLIPB9, and CLIPA14, alongside CLIPB8, participate in prophenoloxidase activation in melanisation process and play roles in the Toll and IMD pathways of the innate immune response in Aedes mosquitoes [76,77]. Our results align with previous study [35], where Dengue virus and Rift valley fever virus infected Ae.…”

Circadian oscillation of perireceptor events influence olfactory sensitivity in diurnal and nocturnal mosquitoes

“…Notable enrichment of CLIP-domain serine protease and serine protease inhibitor genes was observed under the ontology category of serine-type endopeptidase activity ( Figure 6B ; Figure S6B) . Although the function of serine protease and serine protease inhibitors in mosquitoes have mostly been studied with respect to their role in innate immune response ( 55 , 56 ), their physiological relevance is underexplored. It has become increasingly evident that these groups of proteins are widely expressed in brain cells and have a long-term impact on the plasticity, memory, cognition, and behavior of the organism ( 39 , 45 , 57 ).…”

Wolbachia infection-responsive immune genes suppress Plasmodium falciparum infection in Anopheles stephensi

“…berghei ookinete melanization [ 50 – 52 ]. Wang et al (2021) have reported that silencing of CLIPB15 leads to a significant decrease in Phenoloxidase (PO) activity, which acts as a catalyst for the formation of active intermediates of quinone for the synthesis of melanin, but silencing of CLIPB22 does not alter the PO activity [ 48 ]. Silencing of both CLIPB15 and CLIPB22 affects the survival of Ae .…”

“…Silencing of both CLIPB15 and CLIPB22 affects the survival of Ae . aegypti after pathogenic bacterial infection [ 48 ]. Consistent with these studies, we identified three CLIP domain-containing serine proteases, two of which, CLIPB2 and CLIPB4 , were primarily upregulated in Wolbachia -infected blood-fed An .…”