Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan

PLOS Genetics · 15 authors, 6 centres

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FIDELITY 100%

POPULATIONAdult Drosophila melanogaster (wDah, Canton-S strains)

INTERVENTIONBacteria-conditioned diet (BacD) or heat-killed bacteria or purified peptidoglycans from Acetobacter persici Ai or Lactiplantibacillus plantarum Lsi

COMPARISONControl diet (MRS broth only, with antibiotics) or diet conditioned with the other bacterial species

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TL;DR

This study shows that the gut bacterium *Acetobacter persici* shortens lifespan and increases intestinal stem cell proliferation in *Drosophila* through its peptidoglycans, which are sensed by the host receptor PGRP-LC. In contrast, *Lactiplantibacillus plantarum* does not shorten lifespan because its peptidoglycans preferentially activate the negative regulator PGRP-LE, leading to immune tolerance. These findings reveal that specific bacterial cell wall components can shift the trade-off between host defence and longevity, with clinical implications for understanding how gut microbes influence ageing through postbiotic mechanisms.

Full summary

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**Background** The gut microbiota profoundly influences host physiology, stress resistance, and ageing. In *Drosophila melanogaster*, the gut microbiome predominantly consists of *Lactobacillaceae* and *Acetobacteraceae*. Previous work showed that *Acetobacter persici* promotes ageing and shortens fly lifespan, while *Lactiplantibacillus plantarum* has weaker effects. The molecular mechanisms underlying these species-specific effects on lifespan remain unclear. The immune deficiency (Imd) pathway, homologous to mammalian TNF signalling, is a key pathway through which gut bacteria influence host lifespan. This pathway is activated by diaminopimelic acid (DAP)-type peptidoglycans (PGNs) via two pattern recognition receptors: PGRP-LC and PGRP-LE. The authors aimed to elucidate how specific bacterial species differentially activate the Imd pathway and consequently alter host lifespan and physiology.

**Methods** To overcome technical challenges of gnotobiotic fly experiments (high contamination risk during ageing), the authors developed a bacteria-conditioned diet (BacD) approach. Standard fly diet was inoculated with bacterial isolates (A. persici Ai, L. plantarum Lsi, Gluconobacter sp. Gdi, or Leuconostoc sp. Leui) or MRS broth (control) and incubated at 25°C for 24 hours, allowing bacteria to proliferate to approximately 10^4-10^5 CFU/mg food. An antibiotic cocktail was then added to sterilize the diet. Adult flies were reared on standard diet for 2 days post-eclosion, then antibiotic food for 2-3 days to remove resident bacteria, followed by BacD every 2-4 days for lifespan measurement. Lifespan was assessed in female wDah and Canton-S flies, with log-rank tests for statistical comparison. Intestinal stem cell (ISC) proliferation was quantified by counting phospho-histone H3 (PH3)-positive cells in the midgut. Climbing ability was assessed by negative geotaxis assay. Stress resistance was tested using paraquat (10 mM), starvation (1% agar), and oral infection with *Pseudomonas entomophila*. Transcriptomic analysis (3' RNA-seq) was performed on midguts after 24 hours of BacD treatment. Quantitative RT-PCR was used to validate gene expression changes. Genetic experiments used mutants (DreddB118, RelE20, PGRP-LCE12, PGRP-LE112) and RNAi knockdowns with various drivers (esgts, 5966GS, NP1-Gal4). Heat-killed bacteria (100°C for 15-20 min) and purified PGNs were also tested.

**Key Results** 1. A. persici-conditioned diet significantly shortened lifespan in female wDah (p<0.0001) and Canton-S (p<0.0001) flies, while L. plantarum-conditioned diet did not. Gluconobacter sp. Gdi also shortened lifespan (p<0.0001). 2. A. persici BacD increased ISC proliferation in aged guts (Day 30: mean PH3+ cells ~25 vs ~5 in control, p<0.0001) and decreased climbing ability in aged (Day 35) male flies (p<0.05). 3. A. persici BacD increased resistance to 10 mM paraquat (p<0.0001 for females) and oral P. entomophila infection (p<0.0001 for females), but decreased starvation resistance in females (p<0.0001). 4. Transcriptomic analysis revealed that A. persici strongly induced antimicrobial peptides (AMPs) like Diptericin A (DptA), while L. plantarum preferentially induced amidase PGRPs (e.g., PGRP-SC1a). Both inductions were Imd-dependent, as they were abolished in DreddB118 mutants. 5. DptA induction by A. persici was localized to the anterior midgut, while PGRP-SC1a induction by both species was localized to the posterior midgut. The homeobox gene caudal regulated this regional specificity—caudal knockdown increased DptA and suppressed PGRP-SC1a. 6. DptA induction by A. persici was completely dependent on PGRP-LC (abolished in PGRP-LCE12 mutants), while PGRP-SC1a induction by both species was dependent on PGRP-LE (abolished in PGRP-LE112 mutants). 7. Heat-killed A. persici (OD600=40) recapitulated lifespan shortening (p<0.0001), increased ISC proliferation (p<0.01), and induced DptA expression, but did not increase paraquat resistance or decrease starvation resistance. 8. Purified PGNs from A. persici induced both DptA and PGRP-SC1a, while PGNs from L. plantarum induced only PGRP-SC1a, even at 100-fold higher concentration. 9. Knockdown of Rel or PGRP-LC in progenitor cells (esgts) prevented A. persici-induced ISC proliferation. Knockdown of PGRP-LC in enterocytes (NP1ts) also attenuated ISC proliferation. 10. Knockdown of Rel in enterocytes/enteroblasts (5966GS) mitigated lifespan reduction by A. persici BacD. Knockdown of PGRP-LC using 5966GS reduced lifespan shortening from 22% to 17% median decrease, and using esgts from 19% to 7%.

**Clinical Implications** This study demonstrates that specific bacterial cell wall components (peptidoglycans) can drive ageing phenotypes through distinct host receptor engagement, independent of live bacteria or bacterial metabolites. The finding that heat-killed A. persici shortens lifespan and increases ISC proliferation via PGRP-LC, while L. plantarum does not, highlights the importance of PGN structural differences in determining host outcomes. This has implications for understanding 'postbiotic' effects—how dead bacterial cells or their components influence health. The uncoupling of lifespan shortening (mediated by PGNs) from stress resistance (requiring metabolites) suggests that different bacterial factors independently modulate distinct host pathways. Clinically, this work provides a mechanistic framework for how specific gut bacteria might influence ageing and age-related intestinal dysfunction through pattern recognition receptors, potentially informing microbiome-targeted interventions for healthy ageing. The BacD approach also offers a reproducible tool for studying bacteria-host interactions without the complexity of live gnotobiotic systems.

PICO

PPOPULATION

Adult Drosophila melanogaster (wDah, Canton-S strains)

IINTERVENTION

Bacteria-conditioned diet (BacD) or heat-killed bacteria or purified peptidoglycans from Acetobacter persici Ai or Lactiplantibacillus plantarum Lsi

OOUTCOME

Lifespan, intestinal stem cell proliferation (PH3+ cells), climbing ability, resistance to paraquat, starvation resistance, survival after Pseudomonas entomophila infection, and expression of Imd target genes (DptA, PGRP-SC1a)

STUDY TYPE

other

SPECIALTY

geriatrics

SUMMARISED BY

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FIDELITY CHECK

100% · A

ORIGINAL PAPER

PMC10112789

Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan

PLOS Genetics

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ABSTRACT

Abstract Commensal microbes in animals have a profound impact on tissue homeostasis, stress resistance, and ageing. We previously showed in Drosophila melanogaster that Acetobacter persici is a member of the gut microbiota that promotes ageing and shortens fly lifespan. However, the molecular mechanism by which this specific bacterial species changes lifespan and physiology remains unclear. The difficulty in studying longevity using gnotobiotic flies is the high risk of contamination during ageing. To overcome this technical challenge, we used a bacteria-conditioned diet enriched with bacterial products and cell wall components. Here, we demonstrate that an A . persici -conditioned diet shortens lifespan and increases intestinal stem cell (ISC) proliferation. Feeding adult flies a diet conditioned with A . persici , but not with Lactiplantibacillus plantarum , can decrease lifespan but increase resistance to paraquat or oral infection of Pseudomonas entomophila , indicating that the bacterium alters the trade-off between lifespan and host defence. A transcriptomic analysis using fly intestine revealed that A . persici preferably induces antimicrobial peptides (AMPs), while L . plantarum upregulates amidase peptidoglycan recognition proteins (PGRPs). The specific induction of these Imd target genes by peptidoglycans from two bacterial species is due to the stimulation of the receptor PGRP-LC in the anterior midgut for AMPs or PGRP-LE from the posterior midgut for amidase PGRPs. Heat-killed A . persici also shortens lifespan and increases ISC proliferation via PGRP-LC, but it is not sufficient to alter the stress resistance. Our study emphasizes the significance of peptidoglycan specificity in determining the gut bacterial impact on healthspan. It also unveils the postbiotic effect of specific gut bacterial species, which turns flies into a "live fast, die young" lifestyle.

CITATION EXPORT

Taro Onuma, Toshitaka Yamauchi, Hina Kosakamoto et al.. Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan. PLOS Genetics. (2023). https://doi.org/10.1371/journal.pgen.1010709

AUTHORS

15 · 6 CENTRES

Taro Onuma, Toshitaka Yamauchi, Hina Kosakamoto, Hibiki Kadoguchi, Takayuki Kuraishi, Takumi Murakami, Hiroshi Mori, Masayuki Miura, Fumiaki Obata, Gregory P Copenhaver, Ville Hietakangas, Gregory P Copenhaver, Ville Hietakangas, Gregory P Copenhaver, Ville Hietakangas

1Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
2RIKEN Center for Biosystems Dynamics Research, Hyogo, Japan
3Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan
4Department of Informatics, National Institute of Genetics, Shizuoka, Japan
5Laboratory of Molecular Cell Biology and Development, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
6University of Helsinki: Helsingin Yliopisto, FINLAND