Nubbin isoform antagonism governs Drosophila intestinal immune homeostasis
Fig 5
Enterocyte-specific nub-RB overexpression renders flies hypersensitive to infection despite increased AMP levels and enhanced pathogen clearance.
(A-B) Survival curves of mock-infected (dashed lines) or Ecc15 orally infected female and male flies (filled lines), after simultaneous overexpression (yellow) or downregulation of nub-RB (blue) compared to controls (black) in midgut enterocytes using NP1-Gal4ts. Females mock, n = 24–38; females infected, n = 67–109; males mock, n = 20; males infected, n = 24–32. The graphs are representative of two independent experiments. (C-F) Quantification of relative mRNA expression from whole fly extracts, in uninfected flies or following 3 or 24 h post oral Ecc15 infection, with (yellow rectangles) or without (white circles) simultaneous overexpression of nub-RB in midgut enterocytes. Relative levels were normalized to those of uninfected control flies (set to 1). (G-H) Colony forming units from control flies (white circles), flies with overexpression (yellow squares), or downregulation of nub-RB (blue triangles), orally infected with Ecc15-GFP. Serial dilutions were prepared from whole fly extracts and plated at 1 and 6 hpi. After 1 hpi, remaining flies were transferred to regular food without bacteria. (I-J) Box-Whisker plots (min-max, including outliers) of bacterial clearance from 1 to 6 hpi relative to control flies. (K-L) Capillary feeding assay of the denoted genotypes (N = 5). (M) Representative images of flies from the Smurf assay, initiated at 6 hpi with Ecc15 or control food (Mock) followed by flipping flies onto regular fly food supplemented with blue food dye. No smurfs were recorded within the assayed period (up to two weeks post infection). As control, 5% SDS-supplementation of the food resulted in a high penetrance of smurfs within 3 days (note the blue head and thorax). (N) Post-infection time series of the relative midgut expression of nub transcript forms relative to those at 0 hpi, determined by RT-qPCR (N = 3). Flies (w1118) were starved (S) for 2 h prior to infection (I) on an Ecc15-diet until 6 hpi and subsequently transferred to regular food vials for recovery (R). Bars and boxes denote means ± SE and asterisks (*p<0.05; **p<0.01; ***p<0.001).