Wolbachia Regulate MiRNA To Manipulate The Immune Response Of Aedes Aegypti

Wolbachia,as one of the most widely distributed symbiotic bacteria in the world,can activate the host innate immunity,and confer pathogen interference on Aedes aegypti.A growing research reported that Wolbachia activated host immune response have a broad-spectrum antiviral capacity,such as the significant inhibition on replication of dengue virus and Zika virus.However,the molecular mechanism of Wolbachia boosted immune system has not been fully ascertained.Hence,this study aims to investigate the regulation of Wolbachia on the micro RNA(mi RNA)expression profile as well as the effects of Wolbachia induced differentially expressed mi RNAs on the immunity and metabolism of Aedes aegypti cells.In the present study,the mi RNA expression profiles of wild-type Aedes aegypti cells(Aag2)and Aedes aegypti cells infected with w Alb B strain of Wolbachia(W-Aag2)were determined by next-generation sequencing(NGS).A total of 151 known mi RNAs were detected by mi RNA sequencing.Compared with Aag2 cells,there were 25 upregulated mi RNAs and 30 down-regulated mi RNAs in W-Aag2 cells.Using the bioinformatic prediction of target genes for differentially expressed mi RNAs,a total of 11 differentially expressed mi RNAs might target on genes of immune pathways and energy metabolism.Remarkably,Wolbachia down-regulated mi R-34-5p and up-regulated mi R-980-5p have the most binding relationship with the key genes of the immune Toll pathway and oxidative phosphorylation.In order to further explore whether Wolbachia could utilize mi R-34-5p and mi R-980-5p to modulate immune Toll pathway and mitochondrial respiratory chain,the mi RNA function assay was performed by transfecting the sequence specific mi RNA mimics and inhibitors for mi R-34-5p and mi R-980-5p in cell.The results showed that mi R-34-5p can significantly enhance the expression of the negative regulator of Toll pathway,CACT gene.Meanwhile,mi R-34-5p also suppressed the expression of antibacterial peptides DEFD gene,and the respiratory chain complexes Ⅲ subunit(cytochrome b-c1)gene.Moreover,mi R-980-5p can significantly increase the expression of REL1 A gene and the respiratory chain complex Ⅴ β-subunit.The REL1 A is the key positive regulator of Toll pathway,and the complex Ⅴ β-subunit is the key enzyme of ATP synthesis in the oxidative phosphorylation.To detect the role of Wolbachia on the immune pathway and respiratory chain of Aedes aegypti,the quantity of mitochondria and energy metabolites ATP,ADP and AMP was compared between W-Aag2 and Aag2 cell through high performance liquid chromatography and fluorescence imaging analysis.The data revealed that the mitochondrial mass and the amount of ATP and ADP were significantly increased in W-Aag2 cells.It suggested Wolbachia significantly enhance the energy metabolism of Aedes aegypti cells.In addition,we found Wolbachia significantly upregulated the expression of several immune genes of the Toll pathway via fluorescence quantitative PCR.All in all,we proved that Wolbachia could alter the mi RNA expression profile in Aedes aegypti cell.Notably,Wolbachia employs mi R-34-5p and mi R-980-5p to activate Toll pathway and enhance energy production through manipulating the immune genes and mitochondrial respiratory chain complex genes.On the basis of the reported crucial role of Toll pathway on immune defense against dengue virus infection in Aedes aegypti,our data provides a novel biomarker for elucidating the antiviral mechanism underlying Wolbachia activated host immune response and metabolism.The finding also offers a theoretical basis for better utilization of Wolbachia-based mosquito control technology in the prevention and control of mosquito-borne diseases.