Gut and fat body specific mitochondrial dysfunction in Drosophila infection model

Abstract

Mitochondria are gaining recognition on their pronounced role in the organism beyond the classical energy metabolism organelle, instead panning across immunity, signalling and cellular stress responses. One yet to be understood facet of mitochondrial function is how mitochondrial metabolism affects immune response and tissues associated with immune response. To address this need, Drosophila melanogaster (fruit fly) is an excellent model organism in investigating innate immunity with a high conservation across immune response associated tissues and genes. Recent work in our Mitochondrial Immunometabolism-research group at Tampere University with D. melanogaster has shown that manipulating the oxidative phosphorylation (OXPHOS) complex genes in certain tissues can alter the organism’s fitness to combat infection, depending on the nature of mitochondrial interference and target tissues.

In my thesis, the role of mitochondrial OXPHOS metabolism in infection was investigated in the fruit fly model using RNA interference (RNAi) to perturb OXPHOS complex III (cIII) genes’ oxen and UQCR-C1 function in the fat body and gut tissues using the GAL4-UAS system. Furthermore, this perturbation was only introduced after the flies had eclosed and matured using the temporal tubulin-GAL80 temperature-sensitive system (GAL80TS) as a modulator. Previous work and preliminary experiments showed that simply knocking down the OXPHOS cIII genes in the fat body and gut specifically since embryogenesis proved disastrous for the fly, regardless of other tissues being able to normally express genes of interest, highlighting the role of the fat body and gut in organism fitness.

To verify function of the fat body and gut targeted RNAi, RNA was extracted from whole flies, and analysed with RT-qPCR to quantify RNA levels of the gene of interest. This proved problematic due to the primer design and length and overlap of target genes and RNAi sequence. However, a strong phenotype was observed, and correct tissue specific localization was observed with RedStinger label when imaged. Additionally, two new transgenic fly lines were created with the temporal activation Gal80TS component for further dissection of the role of the fat body and midgut separately in infection outcomes, and their function was verified by crossing these newly created fat body-specific and gut-specific driver lines with UAS-RedStinger. The localization of RedStinger expression was correct for both created lines when investigated under red fluorescent light.

Following mitochondrial perturbation in fat body and gut tissues, adult flies were infected with gram-negative bacteria Providencia rettgeri, a natural fly pathogen. Female flies of both genes survived better under the septic injury infection than controls, whereas knockdown males were similar to controls in terms of survival. Sexual dimorphisms in immunity have been observed before, although more often females are reported to be more susceptible to infection than males. Further endeavours to elucidate the role of mitochondrial metabolism in response to infection and outcomes is needed, such as investigating the various metabolic readouts, mode of infection and distinguishing between the role of the fat body and gut in adulthood infection resistance.