Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila

Abstract

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. We report that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP, a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. Our results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut.

Fig 1. Loss of the host receptor for bacteria PGRP-SA or the NF-κB homologue DIF reduces cultivable bacterial density in the gut.

(A) Larvae and 5-day old adults deficient for PGRP-SA have a significantly reduced density of cultivable bacteria while this is not the case for older PGRP-SA^seml mutant flies (30-day old) flies. (B) This was not an effect of the genetic background as 5-day old PGRP-SA^seml mutant flies in a DGRP line 25174 background also displayed reduced cultivable bacterial load. (C) The requirement for PGRP-SA was in enterocytes (ECs) as RNAi of PGRP-SA via the EC-specific NP1-GAL4 driver resulted in a significantly reduced cultivable bacteria load. (D) The Drosophila NF-κB ortholog DIF, downstream of PGRP-SA and the Toll receptor, was also important for the maintenance of bacterial density. (E) Enteric CFU reduction was not dependent on the genetic background since, as their age-matched yw; dif counterparts, the 25174; dif flies had also significantly reduced bacterial density. (F) The yw; dif flies had a reduced lifespan. Each dot represents one gut (n = 15 for both larvae and each adult category) in experiments done in three independent experiments (each experiment with n = 5). The log10 CFU values of mutants and controls were statistically compared using student’s t-test (ns = not significant, *p < 0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Fig 2. Loss of PGRP-SA changes intestinal bacterial composition in young flies.

(A) The graph represents the relative abundance of bacterial families observed in the gut of 5-day and 30-day old female yw, ywseml flies revealed by 16S next-generation sequencing. The x axis represents y strains of different ages, and the y axis represents relative mapped reads. (n = 40 guts/strain). (B) The graphs represent alpha diversity indices of female yw and yw seml across two ages (5 and 30 days). (A) Simpson’s (1-D) index, (B) Shannon H index an (C) Total number of bacterial families observed (Sobs). R represents biological repeat. (n = 40 guts/strain). (C) The graph represents PCA plot (Beta diversity) of female yw and yw seml across two ages (5 and 30 days). R represents biological repeat (n = 40 guts/strain).

Fig 3. In the absence of PGRP-SA, suppression of Drosophila TOR restores intestinal bacterial density.

(A). Silencing of TOR (via RNAi) in enterocytes of male ywPGRP-SA^seml flies or (B) pharmacological inhibition of TOR activity (via administration of rapamycin in the food) restored intestinal bacterial load. For panel A, each dot is an intestine (n = 15 for each category, total of three independent experiments) for panel B, each column is the median of three independent experiments (n = 20 for each experiment). Statistical comparisons were made using student’s t-test (ns = not significant, **p<0.01, ***p<0.001).

Fig 4. Inhibition of Drosophila TOR does not restore bacterial diversity.

(A) The graph represents the relative abundance of bacterial families observed in the guts of 5-day old female yw, ywseml (females) and ywseml; NP1GAL4>UAS-TOR^RNAi flies (males) with (+) or without (-) rapamycin treatment revealed by 16S next-generation sequencing. The x axis represents y strains of different genotypes and treatments, and the y axis represents relative mapped reads. (n = 40 guts/strain/treatment). (B) The graphs represent alpha diversity indices of female yw, ywseml and ywseml; NP1GAL4>UAS-TOR^RNAi across two treatments (with or without rapamycin). (A) Simpson’s (1-D) index, (B) Shannon H index an (C) Total number of bacterial families observed (Sobs). (n = 40 guts/strain/treatment). (C) The graph represents PCA plot (Beta diversity) of female yw, ywseml and ywseml; NP1GAL4>UAS-TOR^RNAi across two treatments (with or without rapamycin; n = 40 guts/strain/treatment).

Fig 5. 4EBP is involved in regulating gut bacterial density as the nexus of Toll/NF-κB and mTOR signalling.

(A). PGRP-SA and DIF influence 4EBP mRNA levels in the Drosophila gut. RT-qPCR analysis of 4E-BP gene expression in the guts of 5-day old female yw seml and yw dif¹ flies relative to their genetic background (yw), which was set to 1 (dotted line; n = 10 guts/strain). (B). Inhibition of mTOR activity restored 4EBP levels in seml mutant guts. RT-qPCR analysis of 4E-BP expression in the guts of 5-day old female yw seml, female yw seml treated with rapamycin and female yw seml; NP1>mTOR^RNAi flies relative to yw female control, which was set to 1 (dotted line; n = 10 guts/strain). (C). 4E-BP mediates rapamycin-induced restoration of cultivable bacterial load in yw seml flies. The graph represents the cultivable gut bacterial load of 5-day old female yw seml (n = 10) and ywseml/Y; NP1>4E-BP^RNAi (n = 10) and ywseml/Y; NP1>4E-BPRNAi + rapamycin (n = 8) flies. In each case (A-C) the x axis represents different fly strains, and the y axis represents relative fold change calculated by ΔΔCT method. n = 3 biological repeats, (ns = not significant, *p<0.05, **p<0.01, ***p <0.001, ****p<0.0001).

Fig 6. Loss of PGRP-SA increases intestinal fat levels.

Loss of PGRP-SA increased intestinal triglyceride levels in 5-day old flies. This phenomenon was suppressed with pharmacological inhibition (rapamycin) or RNAi against TOR in ECs. This was dependent on 4EBP as yw seml; NP1>4E-BP^RNAi treated with rapamycin had fat levels statistically indistinguishable from yw seml. N = 15/genotype/treatment a total of three independent experiments (each with n = 5/genotype/treatment). Values of mutants and controls were statistically compared using student’s t-test (***p<0.001, all other comparisons non-significant except yw seml; NP1>4E-BP^RNAi treated with rapamycin compared to yw, which has a p value<0.001-comparison not shown in the graph).

Fig 7. Reduction of bacterial density is connected to gut lipid catabolism.

Rapamycin treatment or TOR-RNAi in ECs of ywseml flies restored intestinal bacterial CFUs at the levels of the genetic background of the levels of the genetic background of yw. However, this was not the case when the lipase bmm was knocked down in ECs. N = 15/genotype/treatment a total of three independent experiments (each with n = 5/genotype/treatment). Values of mutants and controls were statistically compared using student’s t-test (***p<0.001, NS is non-significant).

Fig 8. RNAi of the Bmm lipase increases intestinal lipid accumulation and reduces bacterial density.

(A) Silencing of bmm expression in enterocytes (through Myo1A^ts-GAL4) resulted in significant reduction of enteric CFUs. (B) This was coupled to an accumulation of lipids in the gut as stained and quantified with Oil Red. Statistical comparisons were conducted using student’s t-test (ns = not significant, ***p<0.001, ****p<0.0001).

Fig 9. Axenic flies show a modest accumulation of enteric lipid levels.

(A) Flies cultured in axenic conditions had on average more Oil red-stained guts. (B) Despite the observed trend this was at the margin of statistical significance (p = 0.15) as calculated using student’s t-test (ns = non-significant).

Fig 10. Bacterial sensing by PGRP-SA is important for the maintenance of intestinal bacterial density.

(A) Mutations (to Alanine) were introduced in three residues spanning the peptidoglycan binding groove. In vitro studies [48] have indicated that S101A increases peptidoglycan (PG) binding, while Y126A and S184A abolish PG binding. (B) Both a wild-type copy of PGRP-SA and PGRP-SA^S101A were able to rescue the significant reduction of CFUs caused by the loss of function PGRP-SA^seml. (C) In contrast, PGRP-SA^Y126A and PGRP-SA^S184A were unable to rescue loss of gut bacterial density. Statistical comparisons were conducted using student’s t-test (ns = not significant, ***p<0.001).

Fig 11. A schematic model outlining the role of PGRP-SA/Toll/Dif in the retention of the gut bacteriome.

PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. This increases 4E-BP transcription/4E-BP protein phosphorylation in enterocytes. 4EBP is important for maintaining normal density of commensal bacteria. We hypothesise that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria.