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Recognition of commensal bacterial peptidoglycans defines Drosophila gut homeostasis and lifespan [1]

['Taro Onuma', 'Department Of Genetics', 'Graduate School Of Pharmaceutical Sciences', 'The University Of Tokyo', 'Tokyo', 'Riken Center For Biosystems Dynamics Research', 'Hyogo', 'Toshitaka Yamauchi', 'Hina Kosakamoto', 'Hibiki Kadoguchi']

Date: 2023-05

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.

Microbiota plays a vital role in our health, but it can also have a negative impact on the lifespan of certain model organisms, such as the fruit fly Drosophila melanogaster. Despite its impact, the molecular mechanism behind how gut bacteria limits host lifespan remains unclear. In this study, we investigated the mechanism that one specific species of microbiota shortens lifespan and disrupts gut homeostasis of the aged flies, using a “fermented” fly diet. We found that the specific effects of this bacterium on fly healthspan were due to its capability of stimulating a specific receptor in the gut that recognizes peptidoglycan, a component of bacterial cell wall. Paradoxically, the same bacterium also increases stress resistance and defence against oral infection of a pathogen. Our study provides insight into the mechanisms underlying how certain members of the microbiota can lead to a “life fast, die young” lifestyle.

Funding: This work was supported by AMED-PRIME to F.O. (JP17gm6010010 and JP20gm6310011), and partly by AMED-Project for Elucidating and Controlling Mechanisms of Aging and Longevity to M.M. (JP21gm5010001). This work was also partially supported by grants from the Japan Society for the Promotion of Science to T.K. (22H02570), and to F.O. (20H05726 and 22H02769), and grants from Japan Science and Technology Agency (JST)-FOREST program to T.K. (JPMJFR2063) and by YakultBio-ScienceFoundation to F. O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Onuma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The magnitude of the immunostimulatory capacity reasonably varies by bacterial species. We have shown that at least three species of Acetobacteraceae strongly activate the Imd pathway and shorten the fly lifespan, while L. plantarum has weaker potency to do so [ 18 , 22 , 23 ]. However, the detailed mechanism by which bacterial factors result in differential levels of Imd activation and hence alter the host lifespan remains elusive. In this study, we described how each gut bacterial species influences the fly physiology, transcriptome, and ageing by using bacteria-conditioned diets (BacDs). We found that bacterial products of A. persici shorten lifespan and increase the resistance to oxidant paraquat and oral infection with the pathogen Pseudomonas entomophila. Contrary to our initial assumption, the predominant mechanism of bacteria-specific effects on ageing and lifespan is not dependent on bacteria-derived metabolites but rather due to sensing the cell wall component PGN by the receptor PGRP-LC. Our data also suggested that A. persici PGN increases host defence against the pathogen but is not sufficient for enhancing resistance to paraquat. This study demonstrates how a gut bacterium shifts the trade-offs between the host defence capacity and lifespan.

In addition, one of the key mechanisms by which the gut bacteria shorten the host lifespan is the immune deficiency (Imd) pathway, which is homologous to the mammalian tumour necrosis factor (TNF) signalling pathway. The Imd pathway has two pattern recognition receptors for diaminopimelic acid (DAP)-type peptidoglycans (PGNs), PGRP-LC and PGRP-LE [ 16 ]. These receptors transmit signals to common downstream factors and eventually activate the transcription factor Relish [ 17 ]. Age-dependent activation of the Imd pathway is attributed to the gut microbiota, which causes age-related dysfunction in various tissues, such as the gut, Malpighian tubules, and brain [ 5 , 18 , 19 ]. Removing the gut microbiota or suppressing the Imd pathway can prevent age-related intestinal dysregulation, which leads to the extension of lifespan [ 20 ]. The majority of age-related transcriptomic alterations are reported to be attenuated in germ-free flies [ 21 ].

Drosophila melanogaster is a powerful model for studying the lifespan-microbiota relationship because of its relatively short lifespan and simple gut microbiome [ 9 ]. The bacterial community in Drosophila predominantly consists of Lactobacillaceae and Acetobaceraceae, including the genera Lactiplantibacillus and Acetobacter [ 10 , 11 ]. Several studies have identified bacteria-derived metabolites that limit lifespan. For example, uracil from Gluconobacter morbifer or Levilactobacillus brevis and lactate from Lactiplantibacillus plantarum are reported to shorten lifespan through reactive oxygen species [ 12 , 13 ]. The gut microbiota also affects two major lifespan-determining mechanisms, the insulin/IGF-1 signalling pathway and the mechanistic target of rapamycin pathway [ 14 , 15 ], although to what extent these pathways contribute to lifespan determination has not been proven.

We live in symbiosis with many microorganisms. The gut microbiome plays an important role in physiology, metabolism, growth, behaviour, and the stress response [ 1 – 3 ]. However, it does not only benefit the host. It has been reported that germ-free animals, including nematodes, flies, and mice, live longer than conventional animals [ 4 – 6 ], especially when nutrients are abundant [ 7 ]. These studies indicate that the gut microbiota can be detrimental to lifespan. The molecular effectors of the gut microbiota on the host healthspan constitute a wide range of metabolites, proteins, and cell wall components [ 8 ]. However, it is not fully understood how each bacterial species in the gut impacts lifespan at the molecular level.

Results

Preparation of a bacteria-conditioned diet We previously isolated A. persici Ai and L. plantarum Lsi as two major bacterial species from the gut of a laboratory strain [23]. We found that A. persici Ai has stronger potency of Imd activation than L. plantarum Lsi, which might be one of the causes of the lifespan-shortening effect of Acetobacteraceae [18]. To further investigate the functional disparity between the two species, we needed to utilize gnotobiotic flies in which the associated microbiome is defined (e.g., monoassociation). Unlike in mice, gnotobiotic experiments in flies are not usually implemented in a “germ-free isolator”. Therefore, one of the difficulties in studying ageing and measuring lifespan using gnotobiotic flies is the higher probability of contamination since their maintenance requires frequently flipping the flies into new vials throughout their lifespan. To make this challenging experiment more technically accessible, we used a bacteria-conditioned diet (BacD), in which bacterial products, as well as bacteria per se, are abundantly present [18]. To prepare the diet, a standard fly diet was inoculated with bacterial isolates (or only the culture medium as a negative control). Incubation of the diet at 25°C for 24 hours allowed the bacterial species to proliferate approximately one hundred-fold (Fig 1A and 1B), reaching 19.1*104 (L. plantarum) or 5.14*104 (A. persici) CFU/mg food that is comparable to the typical bacterial numbers in the standard diet with wild type flies (~104−5 CFU/mg food in case of 1 week old Canton-S male flies) in our laboratory. In addition to the major species, we also investigated two minor bacterial species: Gluconobacter sp. Gdi, a strain of Acetobacteraceae isolated previously from flies with Imd activation [23], and a newly isolated Lactobacillaceae, Leuconostoc sp. Leui (see Methods). PPT PowerPoint slide

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TIFF original image Download: Fig 1. A. persici-conditioned diet shortens lifespan. (A) An overview of preparing the “bacteria conditioned diet (BacD)”. (B) Colony forming units (CFU) of BacD (before addition of antibiotics). For the statistics, one-way ANOVA with Holm-Šídák’s multiple comparison was used. (C) The experimental scheme of the ageing experiments using BacD. (D)(E) Lifespan of female wDah (D) and Canton-S (E) flies with the BacD. A log-rank test was used to compare between control (Ctrl) and each BacD. (F) Phospho-histone H3-positive cell numbers in the midgut of Canton-S female flies fed a conventional diet (left, Day 10 vs Day 41) or 25 days of BacD (right, Day 30). For the statistics, unpaired two-tailed Student’s t test (left figure) or one-way ANOVA with Holm-Šídák’s multiple comparisons (right figure) was used. (G) The climbing ability of Canton-S male flies with either a conventional diet (Left, Day 9 vs Day 22 or Day 41) or BacD (Center, Day 6 or Right, Day 35) assessed by the negative geotaxis assay. For the statistics, unpaired two-tailed Student’s t test (left figure) or one-way ANOVA with Holm-Šídák’s multiple comparison (right figures) was used. The control diet followed the same procedure for BacD but it has only MRS broth in place of bacterial isolates, resulting in the antibiotics-contained diet. Sample sizes (n) and P values are in each figure. https://doi.org/10.1371/journal.pgen.1010709.g001 First, we performed LC–MS/MS analysis to determine how this bacterial conditioning procedure changes the metabolites (i.e., nutrients) levels in the diet. As we previously reported, L. plantarum Lsi-conditioned diets reduced dietary purine levels (S1A Fig, [18]). This is also the case for Leuconostoc sp. Leui. As expected, these two lactic acid bacteria produced lactate (S1A Fig). In contrast, A. persici Ai and Gluconobacter sp. Gdi commonly produced the polyamine spermidine (S1A Fig). These metabolite alterations suggested that BacD can be utilised for understanding how bacteria and bacterial products influence their hosts. Before feeding the diets to the flies, an antibiotics cocktail was added to sterilize the conditioned diet, which prevents further proliferation of the bacteria and stops them from fermenting the diet (Fig 1A). The same antibiotic cocktail was added for the negative control to compare the effect of the bacterial components.

Acetobacter persici Ai-conditioned diet shortened lifespan Experiments using the conditioned diet were conducted using the following scheme. Adult flies were reared with a standard diet for two days post eclosion and then fed with antibiotic food for 2–3 days to remove any resident commensal bacteria in their gut. These flies were then given fresh BacD every 2–4 days to measure their lifespan (Fig 1C). First, we found that the conditioned diet with A. persici Ai or Gluconobacter sp. Gdi significantly shortened the female lifespan of an outbred strain wDah, while that with L. plantarum Lsi or Leuconostoc Leui did not have such a drastic effect (Fig 1D). This was also the case for male flies and the other wild-type strain Canton-S (Figs 1E, S1B, and S1C), indicating a robust phenotype. To test whether an A. persici Ai-conditioned diet promotes ageing, we measured the age-related pathological phenotypes. Accumulating evidence has demonstrated that intestinal stem cells (ISCs) are hyperproliferative in the aged gut, which reflects dysregulation of intestinal homeostasis [10,24]. The increased number of phosphorylated histone H3 (PH3)-positive cells in the aged gut suggested that A. persici Ai induced ISC hyperproliferation (Fig 1F). In contrast, the L. plantarum Lsi-conditioned diet had a milder effect (Fig 1F). We also found that ISC proliferation was promoted by A. persici Ai, not only during ageing but also in the young gut in response to bleomycin, a well-known inducer of DNA damage (S1D and S1E Fig). These data suggested that ISC activity is upregulated by A. persici Ai, which may lead to premature ageing of the gut. Increased ISC proliferation and a shortened lifespan by Acetobacter spp. were also observed in the previous literature [18,22], which further confirmed that BacD could recapitulate at least some of the phenotypes seen in response to live bacteria under a normal laboratory environment. The negative geotaxis assay is widely used to quantify the healthspan of flies [25]. We used male flies to exclude the effect of eggs inside the female body, which affects their climbing speed. As we expected, chronic feeding of the A. persici Ai-conditioned diet but not the L. plantarum Lsi-conditioned diet decreased the climbing ability of the aged (Day 35) flies (Fig 1G). This phenotype was not seen in the young (Day 6) flies (Fig 1G), implying that the bacteria-conditioned diet did not have an acute negative effect on the climbing ability. Together, these data suggested that bacterial products in A. persici Ai promote ageing and decrease the organismal healthspan in both males and females.

Bacteria-conditioned diet did not alter the feeding behaviour Our data indicated that the feeding of BacD with A. persici Ai produced stronger phenotypes than that with L. plantarum Lsi. One hypothesis was that the flies consumed more A. persici Ai-conditioned diet because of the increased appetite, leading to a greater response. To evaluate this possibility, we conducted the capillary feeder assay (CAFE). This assay provides a measure of the flies’ long-term feeding behaviour that quantifies the appetite of the flies. However, no differences were observed in feeding behaviour (Fig 2F). This finding suggests that BacD does not influence the behaviour, and therefore, the differential effect of each BacD on the phenotype of the flies cannot be explained by the amount of food intake.

AMPs and amidase PGRPs were selectively induced via PGRP-LC and LE The most interesting discovery was that only A. persici Ai induced AMP expression even though both A. persici Ai and L. plantarum Lsi have diaminopimelic (DAP)-type PGN. The Imd pathway is redundantly activated via two PGRPs, PGRP-LC and PGRP-LE. To investigate how these receptors contribute to the response to L. plantarum Lsi and A. persici Ai, we tested mutants of Relish, PGRP-LC, and PGRP-LE (RelE20, PGRP-LCE12, PGRP-LE112). As we expected, both DptA and PGRP-SC1a were completely suppressed in RelE20 flies (Fig 5A and 5B). Intriguingly, DptA induction by A. persici Ai was completely suppressed in PGRP-LCE12 flies. On the other hand, in PGRP-LE112 flies, neither L. plantarum Lsi nor A. persici Ai induced PGRP-SC1a (Fig 5A and 5B), indicating that these two receptors regulate different target genes. DptA expression in PGRP-LE mutant flies was highly upregulated in the A. persici Ai-conditioned diet, which suggested that PGRP-LE-dependent induction of amidase PGRPs suppressed DptA induction. Interestingly, PGRP-LE was also expressed in the anterior gut (S3A Fig). Therefore, the differential expression of DptA and PGRP-SC1a in the anterior vs posterior gut is not due to the region-specific expression of PGRP-LC and LE. We also found that DptA was induced only by A. persici Ai in other tissues, such as the thorax and head, which was again suppressed in PGRP-LCE12, but not PGRP-LE112 (S3B and S3C Fig). Taken together, these data demonstrated that A. persici Ai systemically stimulates PGRP-LC, whereas L. plantarum Lsi only stimulates PGRP-LE in the posterior gut. Why L. plantarum Lsi does not activate the Imd pathway through PGRP-LE in the anterior gut is not clear; however, one can assume that the L. plantarum Lsi PGNs may be converted to "active" form (through modification, etc.) that allows it to be recognized by the receptor in the posterior region. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Distinct receptor PGRPs regulate different Imd target genes in gut. (A)(B) Quantitative RT–PCR of the genes DptA (A) and PGRP-SC1a (B) in female wDah, RelE20, PGRP-LCE12, and PGRP-LE112 fly guts after 24 hours of BacD. For the statistics, ANOVA with Holm-Šídák’s multiple comparison was used. The control diet followed the same procedure for BacD but it has only MRS broth in place of bacterial isolates, resulted in the antibiotics-contained diet. Sample sizes (n) and P values are in each figure. https://doi.org/10.1371/journal.pgen.1010709.g005 Since our BacDs includes the antibiotics cocktail, we checked whether side effect of antibitoics would interfere with the phenotypes caused by BacD. The differential induction of Imd target genes as well as the enhanced paraquat resistance or the reduced starvation resistance with A.persici Ai-conditioned diet was observed even in the absence of the antibiotics cocktail (S4A, S4B and S4C Fig).

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010709

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