Differences in proteome perturbations caused by the Wolbachia strain w Au suggest multiple mechanisms of Wolbachia -mediated antiviral activity

Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue virus (DENV) and other RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for DENV control. The degree of virus inhibition varies between Wolbachia strains. Density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains w Au and w AlbA are maintained at similar intracellular densities but only w Au produces strong DENV inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains w Mel/ w MelPop in Ae. aegypti. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines carrying strains w Au and w AlbA: with the hypothesis that differences in perturbations may underline Wolbachia -mediated antiviral activity. Surprisingly, w Au-carrying midguts not only showed distinct proteome perturbations when compared to non- Wolbachia carrying and w AlbA-carrying midguts but also w Mel-carrying midguts. There are changes in RNA processing pathways and upregulation of a specific set of RNA-binding proteins in the w Au-carrying line, including genes with known antiviral activity. Lipid transport and metabolism proteome changes also differ between strains, and we show that strain w Au does not produce the same cholesterol sequestration phenotype as w Mel. Moreover, in contrast to w Mel, w Au antiviral activity was not rescued by cyclodextrin treatment. Together these results suggest that w Au could show unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.