TOR signalling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila

Abstract

When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. Although most attention has focused on the role of innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, we identify induction of intestinal target-of-rapamycin (TOR) kinase signalling as a key adaptive metabolic response to enteric infection. We find that enteric infection induces both local and systemic induction of TOR independently of the Immune deficiency (IMD) innate immune pathway, and we see that TOR functions together with IMD signalling to promote infection survival. These protective effects of TOR signalling are associated with remodelling of host lipid metabolism. Thus, we see that TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. Our data support a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival. This article has an associated First Person interview with the first author of the paper.

Keywords: Drosophila; Infection; Lipid metabolism; Physiology; TOR.

Fig. 1.

Enteric bacterial infection stimulates local and systemic TOR activity. (A,B) Western blots of intestines from adult flies subjected to 4 h oral P. entomophila (P.e.) infection using antibodies to phosphorylated S6K (pS6K) (A), phosphorylated S6 (pS6) (B) and actin (as a loading control). (C,D) Western blots of either whole animals (C) or isolated abdominal samples (D) from adult flies subjected to 4 h oral P. entomophila infection using antibodies to phosphorylated S6K (pS6K), phosphorylated Akt (pAkt) and actin (as a loading control). (E,F) qPCR analysis of the FOXO target gene, 4EBP, from either whole-body samples (E) or abdominal samples (F) of control or P. entomophila-infected flies. Bars represent mean±s.e.m., individual data points are plotted as circles. (G) qPCR analysis of ILP mRNAs from whole-body samples of control or P. entomophila-infected flies. (H) Western blots of whole-body samples from control versus P. entomophila-infected adult flies (genotype: daGAl4 GeneSwitch/+; UAS-ImpL2/+) using antibodies to phosphorylated S6K (pS6K), phosphorylated Akt (pAkt) and actin (as a loading control). ImpL2 induction was achieved by feeding flies RU486 for 3 days before infection (+ RU486). *P<0.05, two-tailed unpaired Student's t-test.

Fig. 2.

TOR and IMD signalling function in parallel to control survival in response to enteric infection. (A-C) Western blots of intestinal (A) or whole-body samples (B, 4 h; C, 24 h) from control versus P. entomophila (P.e.)-infected adult w¹¹¹⁸ or imd mutants using antibodies to phosphorylated S6K and actin (as a loading control). (D) Western blots of intestinal samples from control versus P. entomophila-infected adult w¹¹¹⁸ or Relish mutants using antibodies to phosphorylated S6K and actin (as a loading control). (E,F) Survival plot of control w¹¹¹⁸ (E) and Relish^E20 (rel) mutant (F) mated female flies subjected to 48 h oral P. entomophila infection. Animals were then returned to standard food and the percentage of animals surviving was counted. N≥50 animals per experimental condition. *P<0.05, log rank test.

Fig. 3.

Induction of intestinal TOR signalling is not required for systemic AMP induction. (A-D) qRT-PCR analysis on adult w¹¹¹⁸ mated females subjected to a 24 h pre-treatment of rapamycin or DMSO control followed by 24 h oral P. entomophila (P.e.) feeding along with rapamycin. mRNA transcript levels of anti-microbial peptides (AMPs) are presented as relative changes versus control (corrected for RpS9). (E) Pathogen abundance [in colony forming units (CFUs) per fly] control and rapamycin-treated flies at 4 h and 24 h during the 2 h infection period and at 24 h and 72 h post-infection. The bars represent the mean for each condition, with error bars representing the s.e.m. and individual values plotted as symbols. Two-way ANOVA followed by two-tailed unpaired Student's t-test. ND, no detectable colonies; ns, not significant.

Fig. 4.

TOR signalling is required to limit excess lipid loss following enteric infection. (A) Total triacylglyceride (TAG) levels in control versus 48 h P. entomophila (P.e.)-infected adults. (B) BODIPY staining of fat body of w¹¹¹⁸ control and 24 h P. entomophila-infected mated females. Green, BODIPY. n=5 animals per condition. (C) Lipid droplet accumulation in the anterior region of the intestines stained with Oil- Red O (ORO) from control versus 24 h P. entomophila-infected flies. High levels of lipid accumulation were seen in the anterior regions of control guts (indicated with dashed line) and this was decreased in infected guts (n=10 per condition). (D) ORO intensities in the anterior regions (indicated with dashed lines in C) of w¹¹¹⁸ control and 24 h P. entomophila-infected intestines. (E) BODIPY staining of anterior regions of w¹¹¹⁸ control and 24 h P. entomophila-infected intestines. Green, BODIPY; blue, Hoechst DNA dye. n=5 guts per condition. A representative image is shown. (F) Adult w¹¹¹⁸ mated females subjected to 24 h pre-treatment of rapamycin followed by 24 h oral P. entomophila feeding along with rapamycin. TAG assays were performed on flies at 0, 1 or 3 days after infection. The bars represent percentage change in TAG levels (compared with uninfected control animals), normalized to the protein content for each condition. Data are mean±s.e.m., individual data points are plotted as circles. *P<0.05 (experimental group compared with the control group at the same time point), two-tailed unpaired Student's t-test. Scale bars: 50 µm (B,E); 200 µm (C).

Fig. 5.

Enteric infection leads to increased expression of lipid synthesis genes in the intestine and abdominal fat. (A) qPCR analysis from whole-body samples of control or P. entomophila (P.e.)-infected flies of lipid synthesis genes (FASN1, ACC, DGAT and Lipin) and two transcription factors (SREBP and Mondo) that control the expression of lipid synthesis genes. (B) qPCR analysis of lipid synthesis genes from abdominal samples of control or P. entomophila-infected flies. (C) qPCR analysis of lipid synthesis genes from intestinal samples of control or P. entomophila-infected flies. Bars represent mean±s.e.m., individual data points are plotted as circles. *P<0.05, two-tailed unpaired Student's t-test.

Fig. 6.

TOR is required for the increased expression of lipid synthesis genes induced by enteric infection. qPCR analysis of lipid synthesis genes and the transcription factors SREBP and Mondo in w¹¹¹⁸ flies pre-treated for 24 h with either DMSO (control, grey symbols) or rapamycin (blue symbols), followed by 24 h of either sucrose (control) or 24 h oral P. entomophila (P.e.) feeding. Bars represent mean±s.e.m., individual data points are plotted as circles. *P<0.05, two-way ANOVA followed by two-tailed unpaired Student's t-test.

Fig. 7.

Enteric infection leads to glycogen mobilization in part through TOR activity. (A) Total glycogen levels in control versus 24 h P. entomophila (P.e.)-infected adults. (B) qPCR analysis of glycogen breakdown genes from whole-body samples of control or 24 h P. entomophila-infected flies. (C) w¹¹¹⁸ mated females were pre-treated for 24 h with either DMSO (control) or rapamycin, followed by 24 h of either sucrose (control) or 24 h oral P. entomophila feeding (grey bars). Whole animals were then processed for qRT-PCR analysis of GlyP mRNA. (D) w¹¹¹⁸ mated females were pre-treated for 24 h with either DMSO (control) or rapamycin, followed by 24 h of either sucrose or 24 h oral P. entomophila feeding. Whole animals were then processed for measurement of total glycogen assays. Bars represent mean±s.e.m., individual data points are plotted as circles. *P<0.05, two-tailed unpaired Student's t-test (A,B) or two-way ANOVA followed by a two-tailed unpaired Student's t-test (C,D).