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. 2022 Feb 7;57(3):361-372.e5.
doi: 10.1016/j.devcel.2021.12.016. Epub 2022 Jan 18.

Bacterial peptidoglycan muropeptides benefit mitochondrial homeostasis and animal physiology by acting as ATP synthase agonists

Dong Tian  1 , Min Han  2
Affiliations

Bacterial peptidoglycan muropeptides benefit mitochondrial homeostasis and animal physiology by acting as ATP synthase agonists

Dong Tian et al. Dev Cell. .

Abstract

The symbiotic relationship between commensal microbes and host animals predicts unidentified beneficial impacts of individual bacterial metabolites on animal physiology. Peptidoglycan fragments (muropeptides) from the bacterial cell wall are known for their roles in pathogenicity and for inducing host immune responses. However, the potential beneficial usage of muropeptides from commensal bacteria by the host needs exploration. We identified a striking role for muropeptides in supporting mitochondrial homeostasis, development, and behaviors in Caenorhabditis elegans. We determined that the beneficial molecules are disaccharide muropeptides containing a short AA chain, and they enter intestinal-cell mitochondria to repress oxidative stress. Further analyses indicate that muropeptides execute this role by binding to and promoting the activity of ATP synthase. Therefore, given the exceptional structural conservation of ATP synthase, the role of muropeptides as a rare agonist of the ATP synthase presents a major conceptual modification regarding the impact of bacterial cell metabolites on animal physiology.

Keywords: ATP synthase; ATP synthase agonist; PG fragments; PGN; UPRmt; bacteria cell wall; food avoidance; mitochondrial stress; muropeptides; unfolded protein response.

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Conflict of interest statement

Declaration of interests The University of Colorado has filed a provisional patent application partly based on results from this study (63/231,350).

Figures

Figure 1.
Figure 1.. Peptidoglycan (PG) from E. coli is required for the development and food behavior in C. elegans
(A) List of 4 E. coli PG metabolic genes identified in a screen of the Keio mutant collection (E. coli K-12). DD-Pase, DD-carboxypeptidase and DD-endopeptidase; LD-TPase, LD-transpeptidase (Typas et al., 2011; van Heijenoort, 2011). (B) Bar graph showing worms fed each of 4 PG mutants displayed growth delay as indicated by percentage of worms at the indicated developmental stages. Larval stages were visually determined based on vulval development. n = 53-140. (C) Worms fed with each of 4 PG mutants exhibited food avoidance behavior. Data are presented as the percentage of worms found On or Off the bacterial lawn. 100-200 animals/assay. Data are represented as mean ± SEM from three replicates. (D-F) Bar graphs and representative microscope images showing that both the growth delay (D, n = 49-78) and food avoidance (E and F, 100-200 animals/assay, mean ± SEM from three replicates) phenotypes were suppressed by adding heat-killed wild type K-12 bacteria (HK-WT) or isolated PG.
Figure 2.
Figure 2.. Absence of beneficial PG metabolites induces Mt stress in C. elegans
(A-D) GFP fluorescence images and bar graphs showing that feeding each of the 4 PG mutant E. coli induced Mt stress as indicated by Mt stress/UPRmt reporters (Phsp-6::gfp and Phsp-60::gfp), and nuclear accumulation of DVE-1::GFP in the intestine. n = 31-68 in (B), n = 31-32 in (C), and n = 39-40 in (D). Error bars: mean ± SD. Scale bar, 100 μm for the top and middle panels in (A), 20 μm for the bottom panels in (A). (E and F) GFP fluorescence images and bar graph showing PG mutant diet did not trigger ER stress (Phsp-4::gfp, n = 27-37, mean ± SD) or cytoplasmic stress (Phsp-16.2::gfp, n = 32-36, mean ± SD). Tunicamycin (n = 29) and heat shock (n = 32) were used as positive controls for ER stress and cytoplasmic stress, respectively. Scale bar in (E), 100 μm. (G) GFP images and bar graph showing PG isolated from wild type E. coli suppressed Mt stress (Phsp-6::GFP) induced by feeding each of the 4 PG mutant E. coli (n = 30-37, mean ± SD). Scale bar, 100 μm. (H and I) Bar graphs showing that, in the germ-free CeMM medium, heat-killed E. coli of each of the four PG mutants induced a high-level Mt stress (Phsp-6::GFP) relative to the level by heat-killed wild type E. coli and such induced-Mt stress was effectively suppressed by supplementing either PG isolated from wild-type E. coli or antioxidant NAC. n = 20-25. Representative images are shown in Figure S3E and S3G. (J) Feeding erfK mutant E. coli upregulated the level of SOD-3::GFP and the increase was suppressed by adding PG from wild type E. coli or NAC. (n = 29-30, mean ± SD). The results from tests done in the germ-free CeMM medium is shown in Figure S3I (K-M), Bar graphs and representative microscope images showing that NAC supplementation suppressed growth delay (K, n = 52-61), Mt stress (L, n = 20-23) and food avoidance behavior (M, 100-200 animals/assay, mean ± SEM from 3 replicates). Scale bar in (L), 100 μm. Representative images for (M) is shown in Figure S3J. *** p<0.001, ns: not significant.
Figure 3.
Figure 3.. Beneficial muropeptides contain disaccharides and short peptides
(A) Cartoon diagram of a large PG molecule and the cleavage points of 3 enzymes indicated by black arrows. NagZ: Glucosaminidase; AmiD: Amidase; NAG: 3’ N-acetylglucosamine; NAM: 5’ N-acetylmuramic acid. (B-D) Bar graphs and microscope images showing the effects of treating isolated PG with the indicated enzymes. ProtesaseK (ProK), AmiD or NagZ treated PG failed to suppress the growth delay (B, n = 50-59), Mt stress (C, n = 30-31, mean ± SD) or food avoidance (D, 100-200 animals/assay, mean ± SEM from 3 replicates) phenotypes in animals fed the erfK mutant E. coli. PG and lysozyme (lyso) treated PG suppressed all three phenotypes. (E-G) Muropeptides isolated from lysozyme-treated PG suppressed growth delay (E, n = 30-65), Mt stress (F, n = 30-32, mean ± SD) and food avoidance (G, 100-200 animals/assay, mean ± SEM from 3 replicates). Scale bar in (C) and (F), 100 μm.
Figure 4.
Figure 4.. Muropeptides enter and accumulate in host intestinal mitochondria
(A) FITC labeled PG (FITC-PG) suppressed the growth defect of worms fed erfK mutant E. coli (n = 64-80). (B) Representative fluorescent images showing in vivo co-localization of FITC-muropeptides and MitoTracter in worm mitochondria (arrows). The arrowheat indicates an auto-fluorescent granule. Scale bar: 10μm. (C) Representative images and quantitative data showing FITC-muropeptide accumulation in intestinal cells of C. elegans that were also fed with MitoTracker as a control. Animals were fixed to remove intestinal auto-fluorescence. n = 17-18, mean ± SD. Scale Bar: 100μm. (D) Representative images and quantitative data showing co-localization of FITC-muropeptides and MitoTracker in a subset of isolated Mt (FITC-positive Mt in upper panel). Quantification showing that 34% of total isolated Mt are FITC-positive, 66% are FITC-negative. FITC-positive Mt may be derived from intestinal cells while FITC-negative Mt may represent Mt from non-intestinal cells. Mean ± SEM, n = 336 from 3 replicates. Scale bar: 5μm. (E) Data from an NF-κB activity assay that indicates the interaction between muropeptides from C. elegans Mt lysates and NOD-1 in mammalian cells. Mt lysate from worms fed wild-type E. coli induced higher NF-κB activity than the lysate from worms fed erfK mutant E. coli. Mean ± SEM from 3 replicates. *** p<0.001, ** p<0.01.
Figure 5.
Figure 5.. Muropeptides bind to ATP synthase in the host for the beneficial function
(A-D) RNAi knockdown of 5 PG interactor candidates triggered Mt stress (A and B, n = 33-37, mean ± SD), growth delay (C, n = 49-60) and food avoidance behavior (D, 100-200 animals/assay, mean ± SEM from 3 replicates). (Representative images for D are shown in Figure S5B). Scale bar in (A) is 100 μm. (E) Interaction between PG and ATP-1 detected by in vivo pull-down assays. ATP-1 from worm lysates was bound to PG and the binding increased in a concentration dependent manner. (F) Proteinase-K treatment (to which short peptides on PG are sensitive), but not trypsin treatment (to which the short amino acid peptides on PG are insensitive), eliminated the binding of PG to ATP-1. (G) Trypsin treatment in Step 4 of the PG purification procedure (Figure S3A), which is expected to remove lipoproteins attached to PG, is required for PG to pull down ATP-1 from worm lysates. (H) GFP images and bar graph showing that the level of FITC-muropeptides in intestinal cells was decreased in atp-4 RNAi knockdown animals. Animals were fixed to remove intestinal autofluorescence. n = 31, mean ± SEM. (I-K) Representative images and bar graphs showing the dependence of the beneficial role of PG on ATP synthase. In atp-4 RNAi knockdown animals, addition of PG failed to suppress Mt stress (I, n = 30-34, mean ± SD), growth delay (J, n = 53-62) and food avoidance behavior (K, 100-200 animals/assay, mean ± SEM from 3 replicates) when feeding with erfK mutant E. coli. atp-4i = atp-4 RNAi treatment. Scale bar in (H), 5 μm; in (I), 100 μm. (L-N) Bar graphs showing that growth delay (L, n = 54-62), Mt stress (M, n = 30-34, mean ± SD) and food avoidance behavior (N, 100-200 animals/assay, mean ± SEM from 3 replicates) in worms fed erfK mutant E. coli were partially suppressed by the mai-2(−) mutation, mai-2(xm19). *** p<0.001.
Figure 6.
Figure 6.. A model for the beneficial role of bacterial muropeptides in host animals
(A) E. coli-produced disaccharide muropeptides act as an ATP synthase agonist by entering host Mt and binding to ATP synthase to promote its activity. The increased ATP synthesis promotes Mt homeostasis, host development and food dwelling behavior in C. elegans. (B) Lack of muropeptides (PG mutant E. coli feeding or germ-free condition) inhibits ATP synthase activity and increases ROS production. The decreased ATP synthesis trigger Mt stress, growth delay and food avoidance behavior in C. elegans. Given that bacterial muropeptides are known to enter cells in mammals and the structure of ATP synthase is exceedingly conserved, such a beneficial role of muropeptides is likely conserved in mammals.
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