Fig 1.
Intestinal PGRP-Lc overexpression drives early mortality and intestinal barrier loss.
(A-F) Lifespan curves (A, C and E) and Smurf proportions (B, D and F) for DaGS > UAS-PGRP-Lc (A and B), S106 GS > UAS-PGRP-Lc ((C and D) and TiGS > UAS-PGRP-Lc (E and F) female flies drug induced (RU25, RU50) from early adulthood and uninduced controls (RU0). n > 200 flies/condition, NS = non-Smurf, SMF = Smurf. Log rank test was used for survival data and binomial test for Smurf proportions. (G and H) Normalised mRNA level for PGRP-Lc and Diptericin (Dpt) in dissected whole gut (G) and abdominal carcass samples (H). n = 6 samples, 5 guts/sample and 5 abdomens (gut removed)/sample. Bar graphs show mean ± SEM. Two-way Anova with Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2.
Overexpression of PGRP-Lc and intestinal barrier loss have distinct impacts on microbial growth.
(A-C) Internal bacterial levels assayed by qPCR of the 16S rRNA gene in surface sterilised females flies drug induced (RU50) from early adulthood and uninduced controls (RU0). n = 6 samples with 5 flies/sample. (A) 5966 > UAS-PGRP-Lc non-Smurf flies, (B) TIGS>UAS-PGRP-Lc non-Smurf and Smurf flies and (C) 5966 > UAS-PGRP-Lc flies monoassociated with Acetobacter pasteurianus (AP), Lactiplantibacillus plantarum (LP) or associated with a mix of both species. Boxplots show display the 25-75th percentiles, with the horizontal bar at the median, and whiskers extending from the minimum to maximum points. Two-way Anova with Tukey’s multiple comparisons. **p < 0.01, ***p < 0.001.
Fig 3.
PGRP-Lc overexpression drives early mortality and intestinal barrier loss independent of the microbiota.
(A and B) Lifespan curves (A) and Smurf proportions (B) for axenic 5966 > UAS-PGRP-Lc female flies drug induced (RU50) from early adulthood and uninduced controls (RU0). n > 200 flies/condition, NS = non-Smurf, SMF = Smurf. Log rank test was used for survival data and binomial test for Smurf proportions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4.
PGRP-Lc overexpression impacts intestinal physiology prior to barrier loss.
(A – E) Representative T.U.R.D plate images (A) and analysis of faecal output (B-E) from conventionally reared 5966 > UAS-PGRP-Lc females at 30 days of age drug induced (RU50) from early adulthood and uninduced controls (RU0). (B) Mean lightness, (C) Mean hue, (D) Mean deposit area and (E) Number of deposits. (F – J) Representative T.U.R.D plate images (F) and analysis of fecal output (G-J) from axenic 5966 > UAS-PGRP-Lc females at 30 days of age drug induced (RU50) from early adulthood and uninduced controls (RU0). (G) Mean lightness, (H) Mean hue, (I) Mean deposit area and (J) Number of deposits. n = 10 plates with 10 flies/plate. Boxplots display the 25–75th percentiles, with the horizontal bar at the median, and whiskers extending from the minimum to maximum points. Mann-Whitney U *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5.
PGRP-Lc overexpression suppresses cell junction gene expression and drives stem cell proliferation.
(A & B) Normalised mRNA level for Snakeskin (A, Ssk) and Mesh (B) in dissected whole gut samples from TiGS > UAS-PGRP-Lc non-Smurf and Smurf females fed RU486 and controls. n = 6 samples, 5 guts/sample. (C & D) Gut diameter at days 3 and 7 (C) and pH3 + cell counts at day 7 (D) post RU486 feeding from TiGS > UAS-PGRP-Lc non-Smurf females. (E) pH3 + cell counts at 25 days of age (15 days post RU486 feeding) from TiGS > UAS-PGRP-Lc non-Smurf and Smurf females fed RU486 and controls. N = (F & G) pH3 + cell counts in non-Smurf TiGS > UAS-PGRP-Lc female midguts at day 7 post RU486 and Gemcitabine feeding (F) and Smurf proportions at 15 days post RU486 and Gemcitabine feeding (23 days old) (G).Bar graphs show mean ± SEM. Boxplots display the 25–75th percentiles, with the horizontal bar at the median, and whiskers extending from the minimum to maximum points. Two-way Anova with Tukey’s multiple comparisons (A, B & E). Mann Whitney U (C & D) Kruskal-Wallis with Dunn’s post hoc test (F) Binomial test (G) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 6.
Both Relish and JNK activation drive early mortality and intestinal barrier loss.
(A – D) Lifespan curves (A & C) and Smurf proportions (B & D) for TiGS > UAS-RelVP16 (A & B) and TiGS > UAS-hepCA (C & D) female flies drug induced (RU25 or 50) from early adulthood and uninduced controls (RU0). n > 200 flies/condition, NS = non-Smurf, SMF = Smurf. Log rank test was used for survival data and binomial test for Smurf proportions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.