1 s2.0 S193131282200395X Main

Article
Gut microbial DL-endopeptidase alleviates Crohn’s

disease via the NOD2 pathway
Graphical abstract Authors
Jie Gao, Xinmei Zhao, Shixian Hu, ...,
Rinse K. Weersma, Xiaolong He,
Hongwei Zhou
Correspondence
r.k.weersma@mdl.umcg.nl (R.K.W.),
hxl2027315@smu.edu.cn (X.H.),
biodegradation@gmail.com (H.Z.)
In brief
Gao et al. show that Firmicutes-derived
DL-endopeptidase generates NOD2
ligands in the gut and that it is depleted
globally in CD patients. The gut
microbiota from CD patients with low DL-
endopeptidase activity increased colitis
susceptibility in mice. Importantly,
restoring NOD2 ligands by administering
DL-endopeptidase, DL-endopeptidase-
producing probiotics, or mifamurtide
exerts anti-colitis effects via NOD2.
Highlights
d Firmicutes-derived DL-endopeptidase increases NOD2
ligand levels in the gut
d DL-endopeptidase is depleted in CD patients and negatively

correlates with colitis
d Fecal microbiota of CD patients with low DL-endopeptidase

predispose mice to colitis
d Supplementation with DL-endopeptidase protects mice

against colitis via NOD2
Gao et al., 2022, Cell Host & Microbe 30, 1–15

October 12, 2022 ª 2022 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.chom.2022.08.002 ll
Please cite this article in press as: Gao et al., Gut microbial DL-endopeptidase alleviates Crohn’s disease via the NOD2 pathway, Cell Host & Microbe
(2022), https://doi.org/10.1016/j.chom.2022.08.002
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Article
Gut microbial DL-endopeptidase alleviates
Crohn’s disease via the NOD2 pathway
Jie Gao,1,12 Xinmei Zhao,2,12 Shixian Hu,3,4,12 Zhenhe Huang,1,12 Mengyao Hu,1 Shaoqin Jin,5 Bingyun Lu,6 Kai Sun,7
Zhang Wang,8 Jingyuan Fu,4,9 Rinse K. Weersma,3,* Xiaolong He,1,10,* and Hongwei Zhou1,11,13,*
1Microbiome Medicine Center, Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong
510655, China
2Guangdong Provincial Key Laboratory of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Department of
Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
3Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen 9713 AV, the
Netherlands
4Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen 9713 AV, the Netherlands
5Department of Gastroenterology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China
6Department of Gastroenterology, Shenzhen Hospital, Southern Medical University, Shenzhen, Guangdong 518101, China
7Department of General Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
8Institute of Ecological Sciences, School of Life Sciences, South China Normal University, Guangzhou, Guangdong 510515, China
9Department of Pediatrics, University of Groningen, University Medical Center Groningen, Groningen 9700 RB, the Netherlands
10Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern
Medical University, Guangzhou, Guangdong 510515, China

11State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, Guangdong 510515, China
12These authors contributed equally
13Lead contact
*Correspondence: r.k.weersma@mdl.umcg.nl (R.K.W.), hxl2027315@smu.edu.cn (X.H.), biodegradation@gmail.com (H.Z.)

https://doi.org/10.1016/j.chom.2022.08.002
SUMMARY
The pattern-recognition receptor NOD2 senses bacterial muropeptides to regulate host immunity and main-
tain homeostasis. Loss-of-function mutations in NOD2 are associated with Crohn’s disease (CD), but how the
variations in microbial factors influence NOD2 signaling and host pathology is elusive. We demonstrate that
the Firmicutes peptidoglycan remodeling enzyme, DL-endopeptidase, increased the NOD2 ligand level in the
gut and impacted colitis outcomes. Metagenomic analyses of global cohorts (n = 857) revealed that DL-endo-
peptidase gene abundance decreased globally in CD patients and negatively correlated with colitis. Fecal
microbiota from CD patients with low DL-endopeptidase activity predisposed mice to colitis. Administering
DL-endopeptidase, but not an active site mutant, alleviated colitis via the NOD2 pathway. Therapeutically
restoring NOD2 ligands with a DL-endopeptidase-producing Lactobacillus salivarius strain or mifamurtide,
a clinical analog of muramyl dipeptide, exerted potent anti-colitis effects. Our study suggests that the deple-
tion of DL-endopeptidase contributes to CD pathogenesis through NOD2 signaling, providing a therapeuti-
cally modifiable target.
INTRODUCTION Crohn’s disease (CD), Blau syndrome, asthma, arthritis, and

many other diseases (Hampe et al., 2001; Hugot et al., 2001;
The nucleotide oligomerization domain 2 (NOD2) receptor is Ogura et al., 2001; Philpott et al., 2014). Loss-of-function muta-
a classical pattern-recognition receptor sensing specific micro- tions in NOD2 are the strongest risk factor for CD (Huttenhower
bial peptidoglycan fragments, including muramyl dipeptide et al., 2014; Jostins et al., 2012; Nayar et al., 2021; Sazonovs
(MDP), and N-acetylglucosamine (NAG)-MDP (Caruso et al., et al., 2021). Nearly 50% of familial CD cases in Western
2014; Girardin et al., 2003; Wolf et al., 2016). Activation populations are associated with three mutations: rs2066844,
of NOD2 is involved in a lot of physiological processes, including rs2066845, and rs2066847, all of which impair NOD2 ligand
immune training, epithelial regeneration, insulin resistance, recognition. These studies indicate the critical role of NOD2
vaccination response, and promoting the effects of anti-PD-L1 signaling in mediating host homeostasis.
immunotherapy (Cavallari et al., 2017; Griffin et al., 2021; Kim Aside from receptor defects, whether NOD2 ligand deficiency
et al., 2016; Mulder et al., 2019; Nigro et al., 2014). Impaired could influence NOD2 signaling remains largely unknown. Pepti-
NOD2 sensing caused by gene mutation is associated with doglycan is the main structural component of the bacterial cell
Cell Host & Microbe 30, 1–15, October 12, 2022 ª 2022 The Authors. Published by Elsevier Inc. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Figure 1. Distribution of eight types of PGHs-encoding genes in 1,520 gut-bacterial reference genomes
(A) The scheme of catalytic sites of eight types of PGHs.
(B) Distribution of eight types of PGHs-encoding genes in 1,520 reference bacterial genome. The color of tips indicates the taxonomy of the genome at phylum levels;
the 1–8 circles show the copy number of different types of PGHs-encoding genes; the outmost histogram demonstrates the sum of PGH types encoded by a
genome.
(C) Distribution of eight types of PGHs-encoding genes according to different phylum.
NAM, N-acetylmuramic acid; NAG, N-acetylglucosamine; D-Ala, Dalanine; L-lys, L-lysine; D-Glu-NH2, D-glutamine; L-Ala, Lalanine.
See also Table S1.
wall, and it is estimated that there is almost 10–100 g peptido- that complementary to genetic mutations, gut microbiota dysbio-
glycan in our gut, which could be released as soluble fragments sis could also lead to NOD2 deficiency, which contributes to CD
during bacteria growth and maturation (Laman et al., 2020). pathogenesis.
Peptidoglycan hydrolases (PGHs), a group of enzymes that
could be divided into eight subtypes according to the cleavage RESULTS
sites on the peptidoglycan (Figure 1A), catalyzed the gateway
of peptidoglycan remodeling (Egan et al., 2020; Vollmer et al., DL-endopeptidase-encoding genes were highly specific
2008), thus having the potential to influence the compositions to Firmicutes in the human gut
and abundances of peptidoglycan fragments. Despite the pres- As PGHs are the main enzymes responsible for cleaving
ence of a large amount of peptidoglycan in our gut, whether the peptidoglycan chain to release fragments, we systemically
PGHs are redundant in catalyzing the peptidoglycan remodeling evaluated the distribution of PGHs in human gut microbes
remains unclear. As gut microbiota varied greatly among individ- via the profiling of 8 types of PGH-encoding genes in the genome
uals (He et al., 2018; Lozupone et al., 2012; Yatsunenko et al., of 1,520 gut-bacterial reference genomes (Zou et al., 2019).
2012), it is plausible that a disruption in gut microbiota composi- The results revealed a differential distribution pattern of PGH-en-
tion may lead to PGH perturbation and NOD2 ligand deficiency. coding genes in bacterial genomes: muramidase-, amidase-,
Here, we demonstrated that Firmicutes-derived DL-endopepti- and DD-carboxypeptidase-encoding genes were widely distrib-
dases are critical for NOD2 ligand production. Furthermore, uted across all phyla, whereas genes encoding DL-endopepti-
we found DL-endopeptidase gene abundances decreased dases, especially the secreted DL-endopeptidases, were specif-
universally in CD patients and negatively correlates with colitis ically present in certain bacteria from Firmicutes, particularly
activity. Fecal microbiota from CD patients with low DL-endopep- Erysipelotrichaceae, Lachnospiraceae, and Ruminococcaceae
tidase activity predispose mice to colitis. DL-endopeptidase- (Figures 1 and 2A; Table S1).
based treatments, including a DL-endopeptidase-producing Further quantifying PGH-encoding genes in 2,324 metage-
Lactobacillus salivarius (L. salivarius) strain, or mifamurtide effec- nomic samples from 5 countries revealed that the genes encod-
tively alleviated colitis via NOD2. Together, these results suggest ing muramidase-, amidase-, and DD-carboxypeptidase were the
2 Cell Host & Microbe 30, 1–15, October 12, 2022

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Figure 2. DL-endopeptidase-encoding genes are highly enriched in specific bacteria from Firmicutes, and their abundance vary across pop-
ulations
(A) Distribution of DL-endopeptidase genes in 1,520 bacterial reference genomes. The color of tips indicates the taxonomy of the genome at the phylum level; the
1–4 circles show the copy number of different types of DL-endopeptidase-encoding genes. The lower panel shows the distribution of DL-endopeptidase-en-
coding genes according to conserved domain and cellular location in Firmicutes, Bacteroidetes, and Actinobacteria.
(B) Left panel: relative abundance of eight types of PGH-encoding genes in 11 metagenomic cohorts. Right panel: the variations of four types of PGHs, shown as
relative abundance (the left and right whiskers present the distance between the 25th and 75th percentiles, and the square is the median), and coefficient of
variation (CV%).
(C) Phylogenetic tree analysis of DL-endopeptidase sequences quantified in more than 40% samples in at least one cohort. The outmost circle shows the
conserved domain organization of each sequence; the inner circle shows the taxonomy of each sequence at phylum level. The color of tips indicates the cellular
location of each sequence.
See also Table S1.
dominant PGH-encoding genes in the gut, while there enzyme in the microbiome, while the presence and abundance
was a great variation in the abundance of DL-endopeptidase-en- of DL-endopeptidase-encoding genes vary.
coding genes (coefficient of variation: 34.4%, 20.0%, 32.7%,
and 55.8% for each; Figure 2B). Then, we focused on 57 DL- DL-endopeptidase determines the generation of NOD2
endopeptidase sequences present in more than 40% samples ligands in the gut
in at least one cohort. Again, we found DL-endopeptidases Next, we systemically profiled the NOD2-activating ability of 46
from Firmicutes constituted more than 90% (52/57) of these commensal strains in Firmicutes and Bacteroidetes from our
sequences (Figure 2C). Previous studies found that the combina- culturomics datasets (Figure 3A; Table S2). The results showed
tion of muramidase and DL-endopeptidase could generate that all the strains with NOD2-activating ability encode DL-endo-
NOD2 ligands from intact peptidoglycan (Kim et al., 2019). Our peptidase, with the exception of L. salivarius, which activated
genomic and metagenomic analyses indicated that in the com- NOD2 but without the identification of known DL-endopepti-
plex ecology of the gut, muramidase may be a redundant dase-encoding genes. Further comparative genomic analysis
Cell Host & Microbe 30, 1–15, October 12, 2022 3

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Figure 3. DL-endopeptidase determines the generation of NOD2 ligands in the gut

(A) Heatmap illustrating the NOD2-activating ability of 46 strains, each row represents one strain, with taxonomy information presented on the left; the color of
cells indicates the ability to activate NOD2, and green indicates a strong ability to activate NOD2.
(B) Phylogenetic clustering of NLPC_P60 containing sequences from L. salivarius (UC118), Enterococcus faecium (SagA), Enterococcus faecalis (EFOG), and
Lactobacillus acidophilus (LAN55, LAN98, LAN50, and LAN70).
(C) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the products of different DL-endopeptidases + mutanolysin-digested peptidoglycan. BSA and
MDP were served as negative and positive control, respectively. The values are relative ratios normalized to BSA treatment, valued as 1. n = 3; data represent 3
independent experiments.
(D) Immunoblot analysis of UC118 from the lysate or culture supernatant of L. salivarius.
(E) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the supernatant of E. coli or S. aureus pre-incubated with BSA, UC118, or SagA. The values are
relative ratios normalized to BSA treatment, valued as 1. n = 3; data represent 3 independent experiments.
(F) Mice were gavaged with Cy3-labeled UC118 or pectin/zein complex-coated Cy3-labeled UC118. Colonic sections were prepared to detect the distribution of
UC118 (red). An anti-E-cadherin antibody was used to stain the epithelial cell (green). Scale bars, 200 mm. White arrows indicate the presence of UC118 in the
colonic lumen.
(G) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the fecal supernatant, ileum, or colon homogenate of mice gavaged with indicated doses of
pectin/zein complex-coated muramidase or UC118. The values are relative ratios normalized to BSA-coated pectin/zein beads, valued as 1. n = 6 mice/group;
data represent 2 independent experiments.
(H) Correlation between fecal NOD2-activating ability and DL-endopeptidase-encoding taxonomy abundances in the human samples (left, n = 17). Right indicates
the taxonomy information of these bacteria and the distribution of DL-endopeptidase genes in the corresponding taxa at family level.
MOI, multiplicity of infection. Data are presented as mean ± SD. Statistical comparison was performed by Student’s t test (C), one-way ANOVA followed by
Bonferroni post hoc test (E and G), or Pearson correlation test (H). ** p < 0.01 and *** p < 0.001; ns, not significant.
See also Figures S1 and S2 and Table S2.
in combination with the NOD2 activation assay characterized a W6Y mutation on the 6th position of the NLPC_P60 domain,
a DL-endopeptidase (named UC118 here) from L. salivarius which is critical for its substrate binding (Kim et al., 2019). Phylo-
with full functional domain but with less than 15% identity with genetic analysis showed a separation between sequences from
known DL-endopeptidase sequences (Figures 3B and 3C; see activator and non-activator from the same genus (Figure S1).
STAR Methods). Thus, the presence of DL-endopeptidase-en- Since some DL-endopeptidases are secreted enzymes
coding genes in a bacterial genome indeed is required for its abil- (Figure 2A), we speculated that they could interact with other
ity to shed NOD2 ligands. We also observed that some species bacteria and enable them to release NOD2 ligands. We used
encoding DL-endopeptidase genes failed to activate NOD2 two secreted DL-endopeptidases to verify this speculation:
(Figure 3A). Sequence analysis found that some of them have SagA, a known secreted DL-endopeptidase from Enterococcus

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Figure 4. DL-endopeptidase genes were depleted in CD patients and negatively correlated with colitis activity
(A) A schematic diagram of DL-endopeptidase-NOD2-ligand-signaling axis (left) and the changes of DL-endopeptidase-NOD2 ligands and their association
colitis activity in four cohorts (right). Yellow dots represent DL-endopeptidase gene abundance generated from metagenomic datasets, green dots represent
NOD2 ligands quantified using HEK-Blue-NOD2 cells, and gray dots refer to colitis activity evaluated by immune cell types and clinical parameters. The details of
the data are shown in (B)–(G).
(B) The gene abundance of DL-endopeptidases from Firmicutes in the iHMP-, Spanish, or Chinese IBD cohort according to cellular location and conserved
domain (n = 168, 139, and 29, respectively).
(C) The fecal NOD2-activating ability (upper panel, n = 55) or fecal MDP levels (lower panel, n = 36) in CD or health controls from the Chinese cohort, as detected by
the HEK-Blue-NOD2 cells or HPLC-MS/MS, respectively. The values of fecal NOD2-activating ability are relative ratios normalized to the mean value of health
control, valued as 1.
(D) Correlation between DL-endopeptidase-encoding gene abundances and the transcriptional levels of RIPK2 and ATG16L1, and the expression of genes
specific to macrophages M1/M2, resting mast cells in the ileum tissue from the iHMP cohort (n = 37).
(legend continued on next page)

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faecium (Rangan et al., 2016), and UC118, a secreted DL-endo- quantification with ShortBRED (Figure S3B). Specifically, the
peptidase as described above (Figures 3C and 3D). The results abundance of genes encoding DL-endopeptidases with
showed that adding UC118 or SagA to non-NOD2-ligand- NLPC_P60 or Peptidase_M14 domain from the Firmicutes
releasing bacteria induced more potent NOD2 activation than was significantly decreased in CD patients in three cohorts (Fig-
bovine serum albumin (BSA; Figure 3E). Further, we explored ure 4B). In addition, fecal NOD2-activating ability and MDP levels
whether supplementation with DL-endopeptidase could induce were significantly reduced in CD patients from the Chinese cohort
NOD2 ligand release in the gut in vivo. To avoid the enzymes (Figure 4C).
being destroyed in the gastrointestinal tract, a pectin/zein-based To interrogate the association between DL-endopeptidases
complex delivery system was used to deliver UC118 as and tissue inflammation, we evaluated the immune infiltration
described previously (Yan et al., 2011), and the results showed of ileum tissues from the iHMP transcriptional data using
that UC118 was successfully delivered to the colonic lumen CIBERSORT (Newman et al., 2015) (n = 37; see STAR Methods).
(Figure 3F; see STAR Methods). We found that the feces and Among 22 profiled immune cell types, DL-endopeptidase gene
intestinal tissues from the mice gavaged with DL-endopepti- abundance significantly positively correlated with the expression
dase, in contrast to muramidase, induced a significant NOD2 of genes specific to macrophage M2 and resting mast cells, while
activation in a dose-dependent manner (Figure 3G). Finally, negatively correlated with those of macrophage M1, indicating its
we analyzed the correlation between the fecal NOD2-activating negative association with inflammatory states (Figure 4D;
ability and taxonomy abundance in 17 human samples and Table S4). Meanwhile, DL-endopeptidases showed an inverse
found that all the bacteria that positively associated with NOD2 correlation with ATG16L1 and RIPK2 transcriptional levels (Fig-
activation were DL-endopeptidase-encoding bacteria from ure 4D), both of which are associated with NOD2 downstream
Firmicutes (Figure 3H). To directly analyze the relationship signaling (Magalhaes et al., 2011; Sorbara et al., 2013). One
between DL-endopeptidases and NOD2 ligands in the human possible explanation for this discrepancy is that RIPK2 could be
gut ecology, we performed shotgun metagenomic sequencing up-regulated independent of NOD2 in an inflammatory state
of another 19 human stool microbiomes, and target-quantified (Honjo et al., 2021; Kobayashi et al., 2002; Lu et al., 2005; Usluoglu
DL-endopeptidase gene abundances confirmed that the et al., 2007; Watanabe et al., 2019).
secreted DL-endopeptidases from Firmicutes showed a robust In addition, NOD2 ligand levels and DL-endopeptidase gene
positive correlation with NOD2 activation (Figures S2A and abundances negatively correlated with colitis activity in the Chi-
S2B). Based on these findings, we concluded that DL-endopep- nese CD cohort, where all patients are without NOD2 mutations
tidase determines the NOD2 ligand levels in the gut. (Figure 4E; Table S5). As a validation, in a large Netherlands cohort
encompassing 494 IBD samples, DL-endopeptidase gene abun-
dance was negatively correlated with colitis activity (Figure 4F).
DL-endopeptidase genes were depleted in CD patients
More importantly, low DL-endopeptidase gene abundance was
and negatively correlated with colitis activity
associated with structuring/penetrating phenotype after adjusting
To investigate the functional implications of DL-endopeptidase
for NOD2 mutations, age, antibiotics, and C-reactive protein
in CD, we analyzed four independent and geographically
(CRP) levels in the Netherlands cohort (n = 268, odds ratio = 2.056,
dispersed inflammatory bowel disease (IBD) cohorts (n = 857).
p = 0.03; Figure 4G). Taken together, these results showed that Fir-
Among which, the Chinese CD cohort is a sequenced metage-
micutes-derived DL-endopeptidases were depleted in CD and
nomic dataset (n = 29), and three other independent cohorts
negatively correlated with colitis activity.
are publicly available metagenomic studies from the USA (iHMP,
n = 168), Spain (n = 139), and the Netherlands (n = 494)
(Table S3). The changes of DL-endopeptidase gene abundances Fecal microbiota from patients with low DL-
and NOD2 ligand levels, as well as their interaction with disease endopeptidase activities increased the susceptibility to
phenotypes, were assessed when data were available (Figure 4A). colitis in mice via NOD2
HUMAnN2 analysis showed that the abundance of DL-endopep- To investigate whether DL-endopeptidase deficiency could
tidase-encoding genes was significantly decreased in CD edispose mice to colitis and the role of NOD2 signaling in this pro-
patients, compared with controls in the three independent cohorts cess, we collected feces from healthy controls and CD patients,
from different countries (Figure S3A). The decreased DL-endo- divided them into healthy, high-endopeptidase, or low-DL-endo-
peptidase-encoding genes in CD patients were validated by target peptidase activity groups (indicated as healthy, DL-endoHigh, or
(E) Correlation between fecal NOD2-activating ability or DL-endopeptidase-encoding gene abundances and colitis activity index in the Chinese CD cohort
(n = 55).
(F) DL-endopeptidase-encoding gene abundances in patients with different disease behavior (upper panel, n = 258) or with/without perianal disease (lower panel,
n = 437) in the Netherlands IBD cohort.
(G) Forest plot reporting odds ratio of clinical parameters on risk for structuring/penetrating phenotypes in CD from the Netherlands IBD cohort, calculated using
multivariate logistic regression model. NOD2 mutation refers to carriers of rs2066844, rs2066845, or rs2066847. Low DL-endo refers to DL-endopeptidase gene
abundance below the 25th percentile.
UC, ulcerative colitis; ESR, erythrocyte sedimentation rate; CRP, C-reaction protein; Plt, platelets; Fib, fibrinogen; HCT, hematocrit; Hemo, hemoglobin; B1, non-
stricturing and non-penetrating; B2, stricturing; B3, penetrating; PPI, proton pump inhibitor.
Data are presented as mean ± SD. Statistical comparison was performed by Kruskal-Wallis test, and Steel-Dwass test was used for post hoc analysis when three
groups were indicated (B and F), Student’s t test (C), Pearson correlation test (D and E), or multivariate logistic regression (G). *p < 0.05, **p < 0.01,
and ***p < 0.001.
See also Figure S3 and Tables S3, S4, and S5.

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DL-endoLow in Figure 5A), and transferred the fecal microbiota to by fecal microbiota from DL-endoLow (Figures S5E–S5J). Fecal
antibiotic-treated wild-type (WT) mice or their Nod2 knockout NOD2 ligand levels and Erysipelatoclostridiaceae abundance
littermates (Nod2 / ) individually. After 10 days of fecal microbiota positively associated with colonic Atg16l1 transcription, body
transplantation, colitis was induced in mice by adding 3% dextran weight, and colon length, whereas they were negatively core-
sodium sulfate (DSS). To assess the efficiency of microbiota trans- lated with Col1a1 transcription and gut barrier permeability in
plantation, we collected feces from all the donors and recipient mice received fecal microbiota transplantation (FMT) without
mice before the DSS challenge. As expected, the recipient micro- UC118 treatment (Figure S5K). Overall, these results suggested
biomes shifted away from that of donors, as revealed by the that deficiency in DL-endopeptidase increased susceptibility to
tendency of decreased a diversity upon colonization of mice (Fig- colitis via NOD2 pathway.
ure S4A). Moreover, specific taxa, especially those from Firmi-
cutes, failed to engraft in mice, whereas others such as some Bac- Supplementation of DL-endopeptidases protects
teroidetes and Verrucomicrobiota thrived (Figures S4B and S4C). against colitis in mice via NOD2
On average, 70% of the taxa in donor samples engrafted in recip- We next assessed the therapeutic potential of DL-endopepti-
ient mice, with no differences in engraftment between WT and dase and found that supplementation of DL-endopeptidase
Nod2 / mice (Figures S4D and S4E). Like the donors, we did (UC118), but not its active site mutant (UC118-WCH), could exert
observe a difference in microbiota community among recipient a protective effect against DSS-induced colitis (Figures 6A–6F),
mice in healthy, DL-endoHigh, and DL-endoLow groups (Figure 5B; indicating the enzymatic activity is required for UC118’s colitis-
Figures S4F and S4G). Also, LEfSe analysis showed some DL- protecting ability. To validate that UC118 acts through NOD2
endopeptidase gene-encoding taxa were enriched in healthy signaling, we employed GSK717, a selective NOD2 inhibitor
group of recipients, which is validated by linear mixed-effects (Mulder et al., 2019). The colitis severity was comprehensively
models (Figure 5C; Figure S4H; Table S6). And most importantly, evaluated by the previous index, as well as the tissue repair
the fecal NOD2-activating abilities were decreased in order from (proliferating cell nuclear antigen, PCNA), apoptosis (cleaved
healthy to DL-endoHigh to DL-endoLow mice, similarly to that in caspase-3), and inflammation (TNF-a, IFN-g, IL-17, and IL-6).
the human donors (Figure 5D), suggesting that fecal transplanta- We found that the protective effects of UC118 against colitis
tion from human donors into mice maintained differences in were abrogated when mice were treated with GSK717
NOD2-activating ability. (Figures 6G–6M). We confirmed UC118 acted through NOD2
The colitis severity was evaluated by weight loss, colon length, using a 2,4,6-trinitrobenzene sulfonic acid (TNBS) model com-
gut barrier permeability, tissue damage, and inflammatory cyto- bined with Nod2 / mice (Figure S6). These results indicated
kines. In the WT group, mice inoculated with fecal microbiota that DL-endopeptidase protects mice from colitis through pepti-
from DL-endoLow or DL-endoHigh exhibited more severe colitis doglycan remodeling and NOD2 signaling.
upon DSS insulting than those that received fecal microbiota Furthermore, we found that supplementation with a DL-endo-
from healthy donors, among which mice from the DL-endoLow peptidase-producing L. salivarius strain, as well as mifamurtide,
group had the most severe manifestation. In Nod2 / mice, the a clinically proven synthetic lipophilic analog of MDP (Kansara
colitis severity differences between DL-endoLow and DL-endoHigh et al., 2014), could also exert a protective role against colitis
were not evident, whereas the colitis differences between healthy (Figures 7A–7E). Also, the protective capability of L. salivarius
groups and CD (DL-endoLow/DL-endoHigh) remained (Figures 5E– and mifamurtide against colitis was abolished in GSK717-
5M). These results indicated that NOD2 signaling mediates the litis treated mice (Figures 7A–7E). More importantly, we found a
difference between DL-endoLow and DL-endoHigh and that there dose of mifamurtide (12.5 -mg/g body weight) could exert a
could be some other colitis-promoting factors independent of more potent protective effect against colitis than its equimolar
NOD2 in CD patients, compared with healthy control. We found dose of MDP (1 mg MDP equimolar to 2.5 mg mifamurtide;
that transcriptional levels of Atg16l1, instead of Ripk2, decreased Figures 7F–7J), suggesting that mifamurtide has the potential
in Nod2 / mice, indicating Nod2 may regulate Atg16l1 expres- to be repositioned for CD treatment.
sion in vivo, while Ripk2 could be up-regulated in an inflammatory
environment independent of Nod2 signaling (Figures 5N and 5O). DISCUSSION
This was consistent with the positive correlation between
ATG16L1 and DL-endopeptidase gene abundance in the iHMP NOD2 activation is critical for maintaining host homeostasis
database (Figure 4D). via classic ligand-receptor interactions. Previous studies have
Furthermore, we found that even without the DSS challenge, mainly focused on understanding how the changes of receptor
fecal microbiota from DL-endoLow could induce colitis in mice per se, such as gene polymorphism, epigenetic regulation, and
after 1 month of colonization (Figure S5A). Mice that received post-translational modification, influence the NOD2 activation
fecal microbiota from the pooled DL-endoLow patients had the and relate to diseases (Jiang et al., 2013; Lu et al., 2019; Yang
lowest fecal NOD2 ligand levels (Figure S5B), and we observed et al., 2013). Our study provides a fresh insight into NOD2 inactiva-
a clear separation of microbiota structure between groups tion shifting from its receptor to microbiota-derived ligands.
(Figures S5C and S5D). Fecal microbiota from DL-endoLow Through a comprehensive analysis of the human-gut-bacterial
induced weight loss, colon shortening, gut barrier permeability, reference genomes (1,520 genomes), metagenomic data (2,324
and massive collagen deposition, as evidenced by Masson individuals), culturomics (46 strains), and 16S rRNA sequencing
trichrome staining, and significantly elevated Col1a1 transcrip- (17 human samples), our analysis demonstrated that in the com-
tional levels (Figures S5E–S5H). Notably, supplementation with plex gut microbiota ecology, DL-endopeptidases are critical for
DL-endopeptidase could alleviate the colitis symptoms induced NOD2 ligand generation. These data indicated that variation in

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Figure 5. Mice that received fecal microbiota from CD patients with low DL-endopeptidase activity were more predisposed to colitis
(A) Schematic outline of DL-endopeptidase activity stratification and the fecal microbiota transplantation experiments. Details are shown in the STAR Methods.
(B) First two axes of a principal coordinate analysis (PCoA) of weighted UniFrac distances from donors in health, DL-endoHigh, or DL-endoLow groups. Group
differences were tested by pairwise PERMANOVA. n = 3 for each group.
(C) Selected DL-endopeptidase gene-encoding microbiota from Firmicutes decreased in DL-endoHigh or DL-endoLow groups, compared with the healthy con-
trols, as revealed by t statistics of linear mixed-effects analysis: DL-endopeptidase group (healthy, DL-endoHigh, or DL-endoLow) and NOD2 gene status (WT,
Nod2 / ) as a fixed effect and donor ID as a random effect. Healthy and WT were used as references.
(D) NOD2 activity in HEK-Blue-NOD2 cells after incubation with the fecal supernatant of humans (n = 3/group), or mice (n = 9–12/group) that received human fecal
microbiota from indicated groups. The values are relative ratios normalized to DL-endlow group, valued as 1.
(E–H) Colitis severity of mice in different groups: body weight change (E), colon length (F), and intestinal barrier permeability, indicated by FITC-dextran
permeability (G). Histology manifestation on hematoxylin-eosin (H&E) staining, scale bars represent 100 mm, and histology inflammation score (H).
(I–O) The transcriptional levels of Tnfa (I), Il-6 (J), Ccl2 (K), Cxcl1 (L), Col1a1 (M), Ripk2 (N), and Atg16l1 (O) in colonic tissue of mice (n = 8–11/group).
Each dot indicates one sample. Data are shown as mean ± SD. Statistical comparison was performed by two-way ANOVA followed by Bonferroni post hoc test
(E), one-way ANOVA followed by Bonferroni post hoc test (D and F–O), or Student’s t test (N and O). *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.
See also Figures S4 and S5 and Table S6.
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Figure 6. Therapeutic restoration of DL-endopeptidase activities protects against colitis in mice via NOD2 pathway
(A) Upper panel shows the schematic of UC118 domain organization: the NLPC_P60 is green and active site residues are in red. Lower panel shows the NOD2
activity in HEK-Blue-NOD2 cells after incubation with the products of UC118 or its active sites mutation UC118-WCH with mutanolysin-digested peptidoglycan.
BSA and MDP were served as negative and positive control, respectively. The values are relative ratios normalized to BSA treatment, valued as 1. n = 5; data
represent 3 independent experiments.
(B–F) Experimental colitis in mice was induced by DSS as described in STAR Methods. UC118 or UC118-WCH was orally administrated in a dose of 2 mg/g body
weight. Colitis severity of mice in different groups: body weight change (B), colon length (C), and intestinal barrier permeability, indicated by FITC-dextran
permeability (D). Histology manifestation on H&E staining, scale bars represent 100 mm (E), and histology inflammation score (F).
(G–J) Experimental colitis in mice was induced by DSS as described in STAR Methods. NOD2 inhibitor GSK717 was intraperitoneally injected in a dose of 10 mg/g
body weight. Colitis severity of mice in different groups: body weight change (G), colon length (H), and intestinal barrier permeability, indicated by FITC-dextran
permeability (I). Histology manifestation on H&E staining, scale bars represent 100 mm, and histology inflammation score (J).
(K) Representative PCNA staining of mice colonic sections. The number of proliferating cells per crypt was determined (n = 6/group).
(L) Representative cleaved caspase-3 staining of mice colonic sections. The apoptotic index was calculated as described in STAR Methods (n = 6/group).
(legend continued on next page)

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the gut microbiome composition has a potent influence on NOD2 investigated its functional role and found that activating NOD2
signaling and consequently host homeostasis. could maintain gut homeostasis by regulating the immune
Currently, more than 240 genomic loci have been identified to response (Kobayashi et al., 2005; Philpott et al., 2014; Jiang
be associated with CD, and variants in NOD2 had the strongest et al., 2013), secreting a-defensin to modulate gut microbiota dys-
disease predisposing effect (de Lange et al., 2017; Martin et al., biosis (Kobayashi et al., 2005; Petnicki-Ocwieja et al., 2009), and
2019; Nayar et al., 2021). A number of important CD-predispos- enhancing the gut barrier through stimulating stem cell prolifera-
ing genes, such as ATG16L1, LRRK2, XIAP, and IL-10, among tion (Nigro et al., 2014). Consistently, we found a negative correla-
others, were reported to be involved in NOD2 signaling (Chu tion between DL-endopeptidase and gut inflammatory response.
et al., 2016; Noguchi et al., 2009; Rocha et al., 2015; Schwerd Specifically, in the iHMP cohort, DL-endopeptidase gene abun-
et al., 2017; Sorbara et al., 2013). All of these studies have dance negatively correlates with pro-inflammatory M1 and posi-
focused on host-derived factors leading to NOD2 deficiency, tively correlates with pro-repair M2 macrophages. Accordingly,
while how microbiota variation influences NOD2 signaling DL-endopeptidase supplementation decreased the inflammatory
and CD pathogenesis is unexplored. Here, we found Firmi- cytokines in colitic mice. The shift in macrophage populations
cutes-derived DL-endopeptidase genes, which are critical for from M1 to M2 is linked to decreased inflammatory cytokines,
NOD2-ligand shedding, decreased universally (3 countries and disturbance in this process is associated with CD pathogen-
from different continents, 336 samples) in CD. Previous studies esis (Ha et al., 2020; Jain et al., 2015; Zhang et al., 2019). Whether
showed that a decrease in Firmicutes is the consistent finding DL-endopeptidases exert anti-inflammatory responses through
in most CD patients, mainly attributed to the depletion in Rumi- regulating macrophage populations needs to be further investi-
nococcaceae, Roseburia, and Faecalibacterium genera of the gated. Also, the cell-specific Nod2 knockout mice are needed to
Lachnospiraceae (Caruso et al, 2020; Franzosa et al., 2019; comprehensively evaluate the main cell type that mediated the
Lloyd-Price et al., 2019; Manichanh et al., 2012; Zhou et al., protective effects of DL-endopeptidase.
2018), all of which are DL-endopeptidase-encoding taxa. Our Our results have potential therapeutic implications. As indi-
results provide a fresh mechanistic explanation of these taxon- cated by our animal studies, supplementation with DL-endopep-
omy changes from a functional perspective. We have shown tidase not only restores colitis induced by fecal microbiota from
that mice that received microbiota from DL-endoLow are more CD patients with low DL-endopeptidase activity, but it also
susceptible to colitis than those from DL-endoHigh; and impor- exerts potent beneficial effects on chemically induced colitis,
tantly, this difference is diminished in Nod2 / mice, indicating indicating that the restoration or augmentation of the NOD2
the critical role of NOD2 signaling in this process. Notably, pathway by ligand supplementation is a potential promising ther-
Nod2 / mice do not spontaneously develop colitis (Biswas apy for colitis. Further, we found that supplements with a DL-
et al., 2010; Kobayashi et al., 2005; Pauleau and Murray, 2003; endopeptidase-producing L. salivarius strain, isolated from
Ramanan et al., 2014), but microbiota from DL-endoLow patients healthy human gut at our laboratory, could also exert potent
could induce colitis after transplantation for 1 month, indicating anti-colitis effects, which provides a mechanistic explanation
there may be other pathological bacteria in CD patients. Indeed, for some probiotics alleviating colitis in mice models and clinical
studies have revealed that some species enriched in CD patients settings (Derwa et al., 2017; Ganji-Arjenaki et al., 2018; Holowacz
could promote inflammatory responses. For example, Rumino- et al., 2018; Huang et al., 2021). Importantly, we found that mifa-
coccus gnavus produces an inflammatory polysaccharide and murtide, a synthetic analog of MDP approved in Europe to treat
induces inflammatory cytokine (TNF-a) secretion by dendritic osteosarcoma (Kansara et al, 2014), also exerts potent anti-coli-
cells (Henke et al., 2019), Atopobium parvulum induces pancoli- tis effects in mice. Considering the long-proven safety of the
tis in colitis-susceptible Il10-deficient mice through producing traditional probiotic L. salivarius and the clinically proved safety
hydrogen sulfide (Mottawea et al., 2016), and adherent-invasive of mifamurtide, further studies to explore their clinical effects on
Escherichia coli regulates phagocytes to drive intestinal inflam- CD are promising. The baseline and follow-up DL-endopeptidase
mation through metabolism of propanediol (Viladomiu et al., abundance and the NOD2 gene mutation status should be taken
2021). When NOD2 signaling is intact, it could help the host’s de- into account when carrying out these clinical studies.
fense against these pathologic insults and restore gut homeosta- In conclusion, we showed that bacterial DL-endopeptidase
sis in multiple ways (Caruso et al., 2014; Ramanan et al., 2014; is critical for the regulation of NOD2 signaling in CD pathogen-
Mukherjee and Philpott, 2021). Nonetheless, when NOD2 esis, thus providing a promising strategy for treating CD. Given
signaling is compromised in patients with NOD2 mutations or that NOD2 signaling is implicated in a wide range of physiological
low DL-endopeptidase activity, inflammation could occur. activities, manipulation of DL-endopeptidase may have substan-
Although we have convincingly shown that DL-endopeptidase tial biological and clinical importance.
protects mice against chemically induced colitis in a NOD2-
dependent manner, the specific NOD2 downstream pathways Limitations of the study
need to be further investigated. Since NOD2 mutation was re- While the present study has provided compelling evidence that
ported to be associated with CD in 2001 (Hampe et al., 2001; Hugot reduced DL-endopeptidase could influence NOD2 signaling and
et al., 2001; Ogura et al., 2001), a large number of studies have revealed its critical pathological role in CD, it has also instigated
(M) Levels of cytokines (TNF-a, IFN-g, IL-17, and IL-16) in serum of mice.
Each dot indicates an individual mouse (n = 6 mice/group). Data are shown as mean ± SD. Statistical comparison was performed by one-way (A, C, D, F, and H–M)
or two-way (B and G) ANOVA followed by Bonferroni post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.
See also Figure S6.

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Figure 7. Administration of mifamurtide protects against colitis in mice via NOD2 pathway
(A–E) Experimental colitis in mice was induced by DSS as described in STAR Methods. C57/BL6 mice were treated with mifamurtide (Mi) (12.5 mg/g body weight,
intraperitoneal injection) or L. salivarius (L. sa) (109 colony-forming unit/day, intragastric administration). NOD2 inhibitor GSK717 was intraperitoneally injected in a
dose of 10 mg/g body weight. Colitis severity of mice: body weight change (A), colon length (B), and intestinal barrier permeability, indicated by FITC-dextran
permeability (C). Histology manifestation on H&E staining, scale bars represent 100 mm (D). Histology inflammation score (E).
(F–J) Experimental colitis in mice was induced by DSS as described in STAR Methods. C57/BL6 mice were intraperitoneally injected with Mi in a dose of 12.5 mg/g
body weight or MDP in a dose of 5 mg/g body weight. Colitis severity of mice in different treatments: body weight change (F), colon length (G), and intestinal barrier
permeability, indicated by FITC-dextran permeability (H). Histology manifestation on H&E staining, scale bars represent 100 mm (I). Histology inflammation
score (J).
Each dot indicates an individual mouse (n = 5–6 mice/group). Data are shown as mean ± SD. Data represent 3 independent experiments. Statistical comparison
was performed by one-way (B, C, E, G, H, and J) or two-way (A and F) ANOVA followed by Bonferroni post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not
significant.

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compelling questions to pursue in future investigations. For China (81925026, 82130068, 31900101, and 32170111); Dutch Heart Founda-
example, (1) although four IBD cohorts were included here, the tion IN-CONTROL (CVON2018-27); ERC Consolidator grant (grant agreement
no. 101001678); NWO-Vici grant (VI.C.202.022); the Netherlands Organ-on-
sample size of three cohorts is relatively small (n = 168 for iHMP;
Chip Initiative, an NWO Gravitation project (024.003.001); Natural Science
n = 139 for IBD-Spain; n = 55 for CD-China) and (2) the cutoff of Foundation of Guangdong Province of China (2021A1515010455 and
DL-endopeptidase deficiency, as well as the proportion of CD pa- 2021A1515010429); and Medical Scientific Research Foundation of Guang-
tients suffering from NOD2 inactivation induced by low DL-endo- dong Province, China (A2020104 and A2020124).
peptidase abundance, needs to be more accurately defined
using a larger well-designed human cohort with multi-omics AUTHOR CONTRIBUTIONS
characterization.
Conceptualization, H.Z., X.H., J.G., and R.K.W.; methodology, X.H., J.G., S.H.,
Z.H., and M.H.; clinical samples, K.S., X.Z., B.L., R.K.W., and S.J.; visualiza-
STAR+METHODS tion, X.H., J.G., Z.H., S.H., X.Z., and M.H.; funding acquisition, H.Z., J.G.,
X.H., and J.F.; supervision, H.Z., X.H., J.G., R.K.W., and J.F.; writing – original
Detailed methods are provided in the online version of this paper draft, X.H., Z.H., J.G., S.H., and Z.W.; writing – review & editing, H.Z., X.H.,
and include the following: Z.W., J.G., S.H., J.F., and R.K.W.
d KEY RESOURCES TABLE DECLARATION OF INTERESTS

d RESOURCE AVAILABILITY
H.W.Z. is listed as an inventor on patents related to the anti-colitis effect of the
B Lead contact
isolated L. salivarius and mifamurtide.
B Materials availability
B Data and code availability Received: April 11, 2022
d EXPERIMENTAL MODELS AND SUBJECT DETAILS Revised: July 10, 2022
B Patients and samples preparation Accepted: August 3, 2022
B Animals Published: August 31, 2022
B Cell lines and culture conditions
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
E-cadherin Abcam Cat# ab231303; RRID: AB_562059
Rabbit polyclonal anti-UC118 This paper N/A
Rabbit polyclonal anti-PCNA Proteintech Cat# 10205-2-AP; RRID: AB_2160330
Rabbit polyclonal anti-Cleaved-caspase 3 Cell Signaling Technology Cat# 9661; RRID: AB_2341188
Bacterial and virus strains
BL21 (DE3) chemically competent E. coli TIANGEN Cat# CB105-01
Bacillus cereus Lab stock N/A
Bacillus licheniformis Lab stock N/A
Bacillus subtilis Lab stock N/A
Bacillus thermoamylovorans Lab stock N/A
Bacteroides caccae Lab stock N/A
Bacteroides cellulosilyticus Lab stock N/A
Bacteroides dorei Lab stock N/A
Bacteroides eggerthii Lab stock N/A
Bacteroides fragilis Lab stock N/A
Bacteroides ovatus Lab stock N/A
Bacteroides plebeius Lab stock N/A
Bacteroides stercoris Lab stock N/A
Bacteroides vulgatus 1 Lab stock N/A
Bacteroides vulgatus Lab stock N/A
Blautia obeum strain Lab stock N/A
Clostridium butyricum Lab stock N/A
Enterococcus avium Lab stock N/A
Enterococcus casseliflavus Lab stock N/A
Enterococcus faecalis Lab stock N/A
Enterococcus faecium 1 Lab stock N/A
Enterococcus faecium 2 Lab stock N/A
Enterococcus hirae Lab stock N/A
Escherichia coli ATCC Cat# ATCC 25922
Lactobacillus acidophilus Lab stock N/A
Lactobacillus delbrueckii Lab stock N/A
Lactobacillus gasseri Lab stock N/A
Lactobacillus oris Lab stock N/A
Lactobacillus paracasei Lab stock N/A
Lactobacillus plantarum Lab stock N/A
Lactobacillus rhamnosus Lab stock N/A
Lactobacillus salivarius Lab stock N/A
Lactobacillus vaginalis Lab stock N/A
Parabacteroides distasonis Lab stock N/A
Parabacteroides gordonii Lab stock N/A
Parabacteroides merdae Lab stock N/A
Ruminococcus gnavus Lab stock N/A
Staphylococcus aureus 1 ATCC Cat# ATCC 25923
Staphylococcus aureus 2 Lab stock N/A
Streptococcus anginosus Lab stock N/A
(Continued on next page)
e1 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

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Continued
Streptococcus cristatus Lab stock N/A
Streptococcus gallolyticus Lab stock N/A
Streptococcus gordonii Lab stock N/A
Streptococcus mitis Lab stock N/A
Streptococcus parasanguinis Lab stock N/A
Streptococcus pasteurianus Lab stock N/A
Streptococcus salivarius Lab stock N/A
Biological samples
Fecal samples Zhujiang Hospital N/A
Blood samples Zhujiang Hospital N/A
Chemicals, peptides, and recombinant proteins
2,4,6-trinitrobenzenesulfonic acid Sigma-Aldrich Cat# P2297; CAS:2508-19-2
Dextran sodium sulfate Aladdin Cat# D12235; CAS: 9011-18-1
FITC-dextran Sigma-Aldrich Cat# FD40S; CAS: 60842-46-8
Bovine serum albumin Sigma-Aldrich Cat# V900933; CAS: 9048-46-8
Difco Skim Milk BD biosciences Cat# BD-232100
Polyvinylidene difluoride membrane Sigma-Aldrich Cat# 3010040001
Blocking goat serum Solarbio Cat# SL038
Citrate buffer solution,pH 6.0，103 Sigma-Aldrich Cat# C9999
MDP Sigma-Aldrich Cat# A9519; CAS: 53678-77-6
GSK717 Med Chem Express Cat# HY-136555
Dulbecco eagle medium, high glucose GIBCO Cat# 11965092
Heat inactivated fetal bovine serum (FBS) GIBCO Cat# 10100147
Brain Heart Infusion Huankai Microbial Cat# 028361
PYG Broth Huankai Microbial Cat# 027340
Kanamycin sulfate Sigma-Aldrich Cat# 420311; CAS: 25389-94-0
DAPI Sigma-Aldrich Cat# D9542; CAS: 28718-90-3
Isopropyl-b-d-thiogalactoside Sigma-Aldrich Cat# I6758; CAS: 367-93-1
Peptidoglycan from S. aureus Sigma-Aldrich Cat# 77140
Muramidase Sigma-Aldrich Cat# L4919
EcoRI Takara Cat# 1040A
XhoI Takara Cat# 1094A
Sac I Takara Cat# 1078A
BamH I Takara Cat# 1010A
DMSO Sigma-Aldrich Cat# D2650; CAS: 67-68-5
Glycerin Solarbio G8190
Mutanolysin Sigma-Aldrich Cat# M9901; CAS: 55466-22-3
Syrings Filters (0.22 mm) NEST Cat# 331011
L-Glutamine Solution Sigma-Aldrich Cat# 59202C; CAS: 56-85-9
Normocin InvivoGen Cat# ant-nr-1
Blasticidin InvivoGen Cat# ant-bl-05
Zeocin InvivoGen Cat# ant-zn-05
Pectin Aladdin Cat# P112756
HEK-Blue Detection InvivoGen Cat# hb-det2
Zein Aladdin Cat# Z304904; CAS: 9010-66-6
Calcium chloride Sigma-Aldrich Cat# 499609; CAS: 10043-52-4
Hematoxylin-Eosin Solarbio Cat# G1120
5-kDa MWCO columns Sigma-Aldrich Cat# CLS431477
Hypersil GOLD C18 column Thermo Scientific Cat# 25005-059070A
Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022 e2

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Continued
Acquity UPLC HSS T3 column Waters Cat# 186003539
Critical commercial assays
Cy3 Conjugation Kit Abcam Cat# ab188287
His Tagged Protein Purification Kit CWBIO Cat# CW0894
Detoxi-Gel Endotoxin Removing Gel ThermoFisher Cat# 20344
E.Z.N.A. soil DNA Kit Omega Bio-tek Cat# D5625-01
AxyPrep DNA Gel Extraction Kit Axygen Biosciences Cat# AP-GX-250
RNAprep pure Tissue Kit TIANGEN DP431
PrimeScript RT reagent Kit TAKARA Cat# RR037A
(Perfect Real Time)
TB Green Premix Ex Taq II TAKARA Cat# RR820A
(Tli RNaseH Plus)
Imject Freund’s Complete Adjuvant Thermo Fisher Cat# 77140
Imject Freund’s Incomplete Adjuvant Thermo Fisher Cat# 77145
Pierce Protein A IgG Purification Kit ThermoFisher Cat# 44667
Enhanced chemiluminescence reagent kit Bio-Rad Cat# 1705061
Multiplexed Fluorescent Reagent Kit v2-Hs Advanced Cell Diagnostics Cat# 323135
Serum Circulating DNA Kit TIANGEN Cat# DP339
QIAamp DNA Stool Mini Kit QIAGEN Cat# 51504
Mouse TNF-aELISA Kit CUSABIO Cat# CSB-E04741m
Mouse IFN-g ELISA Kit CUSABIO Cat# CSB-E04578m
Mouse IL-17 ELISA Kit CUSABIO Cat# CSB-E04608m
Mouse IL-6 ELISA Kit CUSABIO Cat# CSB-E04639m
Experimental models: Cell lines
HEK-Blue-hNOD2 cells InvivoGen Cat# hkb-hnod2
HEK-Blue Null2 Cells InvivoGen Cat# hkb-null2
Experimental models: Organisms/strains
Mouse: C57BL/6 Southern Medical University N/A
Mouse: C57BL/6-Nod2tm1cyagen Cyagen N/A
New Zealand Rabbits Southern Medical University N/A
Oligonucleotides
Primers for genotyping, see Table S8 This paper N/A
Primers for 16S-rRNA This paper N/A
analysis, see Table S8
Primers for q-PCR, see Table S8 This paper N/A
Recombinant DNA
Plasmid pET28a Merck Cat# 69864
Software and algorithms
ImageJ NIH https://imagej.nih.gov/ij/
R (version 3.6.0) R foundation https://cran.r-project.org
PSORTb (version 3.0) PSORTb https://www.psort.org/
CD-hit (version 4.8.1) (Fu et al., 2012) http://weizhongli-lab.org/cd-hit/
BLASTP (version 2.2.29+) NCBI https://blast.ncbi.nlm.nih.gov
PFAM (version 34.0) (Finn et al., 2016) http://pfam.xfam.org/
Clustal Omega EMBL-EBI https://www.ebi.ac.uk/Tools/msa/clustalo/
Mafft (version 7.450) EMBL-EBI https://www.ebi.ac.uk/Tools/msa/mafft/
FastTree (version 2.1.10) (Price et al., 2009) http://www.microbesonline.org/fasttree/
Kneaddata (version 0.5.1) The Huttenhower Lab http://huttenhower.sph.harvard.edu/kneaddata
ShortBred (version 0.9.5) The Huttenhower Lab http://huttenhower.sph.harvard.edu/shortbred

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Continued
HUMAnN2 Franzosa et al., 2018 https://huttenhower.sph.harvard.edu/humann2/
CIBERSORT Newman et al., 2015 http://cibersort.stanford.edu/
Deposited data
IBD-Spain raw metagenomics EMBL-EBI EMBL-EBI: ERP002061
IHMP raw metagenomics HMP https://ibdmdb.org
IBD-Netherlands raw metagenomics EGA EGA: EGAS00001002702
CD-China raw metagenomics and 16S rRNA sequencing data EMBL-EBI EMBL-EBI: PRJEB51945
CRC-Austria raw metagenomics EMBL-EBI EMBL-EBI: ERP008729
CRC-Germany raw metagenomics EMBL-EBI EMBL-EBI: PRJEB27928
CRC-China raw metagenomics EMBL-EBI EMBL-EBI: PRJEB10878
IBS-USA raw metagenomics EMBL-EBI EMBL-EBI: PRJEB37924
IBS-China raw metagenomics CNGBdb CNGBdb: CNP0000334
Cirrhosis-China raw metagenomics EMBL-EBI EMBL-EBI: PRJEB6337
NAFLD-Europe raw metagenomics EMBL-EBI EMBL-EBI: PRJEB14215
ACVD-China raw metagenomics EMBL-EBI EMBL-EBI: PRJEB21528
PKD-Germany raw metagenomics EMBL-EBI EMBL-EBI: PRJEB17784
Human NOD2 gene polymorphism data NCBI NCBI: PRJNA739398
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hongwei
Zhou (biodegradation@gmail.com).
Materials availability
This study did not generate new unique reagents.
Data and code availability

d The metagenomic and 16S rRNA sequencing data have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI
under accession number PRJEB51945. The human NOD2 gene polymorphism data are available at NCBI under accession
PRJNA739398. All these data are publicly available as of the date of publication. Accession numbers are listed in the key re-
sources table.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODELS AND SUBJECT DETAILS
Patients and samples preparation

The China-CD cohort included 55 individuals (CD = 29, Control = 26, Table S3). The inclusion criteria are: 1) Males or females at 18-60
years old; 2) Patient with the biopsy-proven CD, in the active phase; 3) The age- and sex-matched health controls for each patient. We
excluded patients who used antibiotics or probiotics within the last 2 weeks. The baseline demographic and clinical information were
collected, stool was collected in stool nucleic acid collection and aliquots of stool samples were stored at -80 C until use. DNA was
extracted from aliquots of fecal samples using the QIAamp DNA Stool Mini Kit following manufacturer’s instructions, and then un-
derwent shotgun metagenomic sequencing as described below. Aliquots of fecal samples were examined for NOD2-activating ability
or MDP levels, by the HEK-Blue-NOD2 cells or HPLC-MS/MS respectively. All participants gave written informed consent and the
Medical Ethics Committee of Jiangmen Central Hospital approved the study procedures (ID: Jiangmen Central Hospital [2020]7N).
The Netherlands-IBD cohort made up of 494 patients diagnosed in the specialised IBD clinic of the University Medical Center Gro-
ningen (Groningen, the Netherlands). All of the biopsy-proven IBD patients were included and those didn’t provide fecal samples
were excluded from this study. The mean age of included patients was 43 years old and 39% of them were male. The data collection
and metagenomic sequencing have been described previously (Hu et al., 2021). The Netherlands-IBD cohort was approved by Uni-
versity Medical Centre Groningen IRB (IRB-number 2008.338).

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Animals
C57BL/6 mice (female) and New Zealand rabbits (female) were obtained from the Animal Experimental Center of Southern Medical
University. Nod2 knockout (Nod2tm1cyagen, Nod2-/-) mice, on the background C57BL/6, were obtained from Cyagen Biosciences.
Nod2 heterozygous mice were bred to generate littermate Nod2-/- and Nod2+/+ (WT) mice for experiments. Six-eight weeks old
mice are randomly grouped for experiments. Outcomes of mice were blindly collected. All the animals were raised in specific path-
ogen free conditions with a strict 12 hours light/12 dark shift, access to sterilized water and food ad libitum. The ethics committee of
Southern Medical University approved the animal protocols and animal care and experiments were done strictly adherence to the
Southern Medical University animal care guidelines.
Cell lines and culture conditions

Human NOD2/NF-kB/secreted embryonic alkaline phosphatase (SEAP) reporter HEK293 (HEK-Blue-hNOD2) cells and un-trans-
fected parental HEK-Blue-Null2 cells were obtained from Invivogene and used to detect the activation of NOD2 as described previ-
ously (Huang et al., 2019). Cells were growth in Dulbecco’s modified Eagle’s medium (4.5 g/l glucose), adding 10% (v/v) fetal bovine
serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml NormocinTM, and keeping at 37 C in 5% CO2,
100% humidity. Before the NOD2 activation experiments, cells were maintained for 3 days in a growth medium supplemented
with: Zeocin (100 mg/ml) for Null cells, and Blasticidin (30 mg/ml) for NOD2 cells.
Bacterial strains and culture conditions

All strains from our culturomics and their culture conditions were listed in Tables S2. The culture mediums were pre-reduced in the
anaerobic chamber for at least 48 h. All the anaerobic bacterial strains were grown anaerobically in an anaerobic chamber at 37 C in
peptone-yeast extract-glucose broth under an atmosphere of 5% CO2, 15% H2 and 85% N2. Bacteria were first restreaked from
frozen glycerol stock onto the agar plates. A single colony was then picked and incubated into 5 ml broth. All the aerobic strains
were grown aerobically at 37 C in brain-heart infusion shaking at 200 rpm. Escherichia coli (E. coli, ATCC25922) and Staphylococcus
aureus (S. aureus, ATCC 25923) were obtained from American Type Culture Collection and grown in brain-heart infusion broth at
37 C with agitation (200 rpm).
METHOD DETAILS
Sequence search and protein bioinformatic analysis

To obtain a comprehensive database of PGHs-encoding sequences, we downloaded amino acid sequences that matched the key-
words listed in Table S7 from UniRef90, and the taxonomy was restricted to ‘‘bacteria’’. All the results were manually inspected to
exclude mismatches or fragment sequences.
Taxonomic information regarding these proteins was directly extracted from the FASTA name. The conserved domains of proteins
were predicted using the tool PFAM with default parameter: http://pfam.xfam.org/. The protein’s subcellular localization predictions
were done with PSORTb v.3.0. Sequences similarities comparisons were calculated with Clustal Omega: https://www.ebi.ac.uk/
Tools/msa/clustalo/.
Profiling PGH-encoding genes in 1520 gut reference genomes

The protein-coding sequences of 1520 reference genomes from cultivated human gut bacteria were downloaded from NCBI (Zou
et al., 2019). The PGH sequences retrieved from UniRef90 were searched against the protein-coding sequences of the 1520 refer-
ence genomes using BlastP with a cut-off of identity > 80%, coverage > 80% and e-value < 1e-5.
Public metagenomic datasets and quantification of PGH-encoding genes

In addition to a China-CD cohort (n = 55) and a Netherlands-IBD cohort (n = 494) carried by our team, we also employed another two
publicly available IBD cohorts from Spain (n = 139) or USA (IHMP, n = 168) respectively. Furthermore, night cohorts covering 2047
metagenomic samples from six different countries encompassing eight non-IBD diseases were also included (Bedarf et al., 2017;
Feng et al., 2015; Hoyles et al., 2018; Jie et al., 2017; Lloyd-Price et al., 2019; Mars et al., 2020; Nielsen et al., 2014; Qin et al.,
2014; Wirbel et al., 2019; Yu et al., 2017; Zhao et al., 2020). All the human cohorts have been described in Table S3.
Two methods are employed to quantify PGH-encoding gene abundance in human metagenomic datasets. Method 1: After quality
control, HUMAnN2 (Franzosa et al., 2018) was used to quantify all gene abundance in the UniRef 90 database. The abundance of
PGH-encoding genes was calculated and summarized according to UniRef 90 IDs. Method 2: ShortBRED (Kaminski et al., 2015)
(Version 0.9.5) was employed to target quantify PGH -encoding genes. Firstly, short sequences markers of PGHs families were iden-
tified using UniRef90 database with default parameters, and then quantified these markers in the metagenomic samples, normalizing
by the number of RPKM.
Comparative genomic analysis

L. salivarius could activate NOD2 in our assay (Figure 3A), while blast search didn’t identity known DL-endopeptidase sequence
(identity > 80%, coverage > 80% and e-value < 1e-5). To avoid bias introduced by blast search, we used Hidden Markov
Model search for all the NLPC_P60 containing domains from two pairs of species: Enterococcus faecium (NOD2 activating

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Article OPEN ACCESS
bacteria)/Enterococcus faecalis (NOD2 non-activating bacteria); L. salivarius (NOD2 activating bacteria) and Lactobacillus acidoph-
ilus (NOD2 non-activating bacteria), and 7 sequences were identified (Figure 3B): SagA from Enterococcus faecium (serves as pos-
itive control), EFOG from Enterococcus faecalis, UC118 from L. salivarius and LAN55, LAN98, LAN50, LAN70 from Lactobacillus ac-
idophilus. Phylogenetic Analysis showed that UC118 clustered with SagA, a validated DL-endopeptidase from Enterococcus
faecium, instead of with the NLPC_P60 containing proteins from Lactobacillus acidophilus. Further, these sequences were cloned
into pET28a and proteins were purified respectively as described below.
Fecal 16S rRNA microbial analysis

Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) was entrusted to perform the 16S rRNA microbial analysis of mice fecal
samples. Firstly, E.Z.N.A. soil DNA Kit was employed to isolate fecal DNA. The primers described in Table S8 were used to amplify
16S rRNA genes and the PCR reactions were performed in triplicate. The AxyPrep DNA Gel Extraction Kit was used to purify PCR
products, which were then paired-end sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego,USA) and quantified
with Quantus Fluorometer (Promega, USA). The resulting raw data were demultiplexed and quality-filtered by Fastp (v0.20.0). Demul-
tiplexed sequencing outputs were obtained from the Majorbio Bio-Pharm sequencing facility and analyzed using the QIIME 2 (ver-
sions 2017.9 and 2018.8) software package according to the suggested workflow.
Alpha diversities were analyzed using the Chao index calculated with observed OTUs. Beta diversity was assessed using weighted
UniFrac distance. PCoAs were visualized by R packages ‘‘ggalt’’. Taxonomic analysis was performed using the q2-feature-classifier
trained on GreenGenes OTU table. Differential abundance analysis was performed using the LEfSe. To further analyze the contribu-
tions of DL-endo groups or NOD2 mutation status on each tax feature, a linear mixed effects analysis with DL-endo groups and NOD2
status as a fixed effect and donor ID as a random effect. feature (intercept)+ DL-endo groups+ NOD2 status+(1|donor-ids), healthy
groups and WT mice were set as the controls.
Metagenomic sequence preprocessing

Metagenomic sequencing was performed for fecal samples from China-CD cohort, using the Illumina MiSeq platform. Reads
belonging to the human genome were removed by mapping the data to the human reference genome (version NCBI37). Low-quality
reads were removed using KneadData (V.0.5.1). The subsequent functional quantification was done as described above.
Public transcriptional data and analysis

For transcriptional data in the iHMP cohort, we include only ileum samples taken at the same visit between fecal (metagenomes) and
tissue (transcriptomes). The metadata and gene transcriptional levels, were directly extracted from the data sets. The immune infil-
tration of the samples was evaluated using CIBERSORT methods combined with the LM22 feature matrix (Newman et al., 2015). The
correlation between metagenomics (DL-endopeptidases) and transcription was analysed by Pearson Correlations.
All the bioinformatics tools used in this study were displayed in the key resources table.
NOD2 polymorphisms testing

This study was designed to investigate the NOD2 polymorphisms associated with ligands detection. The genomic DNA in serum
samples from CD patients was isolated using the Serum Circulating DNA Kit (TIANGEN) according to the supplier’s guidelines.
Primers flanking the variants were used for PCR amplification (Table S8). After purification, PCR products were analyzed with the
ABI PRISM Dye Terminator Cycle Sequencing KIT (Applied Biosystems, Darmstadt, Germany) on an ABI 3730xl DNA analyzer using
the same primers applied for amplification. Finally, the sequences were aligned with the human NOD2 coding sequences in the
GenBank database to identify mutations.
NOD2 polymorphisms analyzed in this study are as follows: R38M, A105T, D113N, R138Q, V162I, T189M, L248R, W355., D357A,
I363F, D379A, P463A, L550V, R702W, P727C, A755V, E778K, R790W, N825K, A849V, W907R, G908R, fs1007, L1007P, R1019.
These variants have been reported to associate with ligands sensing (Parkhouse and Monie, 2015).
MDP quantification with liquid chromatography with tandem mass spectrometry (LC-MS/MS)
The MDP levels in fecal samples were quantified by a targeted metabolomic method. Feces were weighed and added with high-
grade water (MOmwas, 500 ml water per 100 mg feces), and mixed by vortexing at max speed for 5 minutes. A sonication bath
was carried out at 25 C for 10 minutes. The supernatant was obtained by centrifuging the mixture at 20, 000 g for 10 minutes
and filtered through a 0.22 mm pore size filter. The flow-through was used for LC-MS/MS analysis. Standard curves were prepared
in high-grade water in a concentration from 12.8 ng/ml to 1600 ng/ml. One hundred microliter samples were separated on a Prelude
SPLC System using Waters Acquity UPLC HSS T3 column (2.1 3 100 mm, 1.8 mm) at 40 C. The mobile phases used as follows: (A)
0.1% FA in water, (B) 0.1% FA in acetonitrile, with gradient of 0 min (2% B), 0.5 min (30% B), 3.5 min (85% B), 5 min (85% B), 6 min
(2% B) at flow rate 0.25 ml/min. Multiple reaction monitoring scanning modes were used for MS analysis (TSQ Quantiva triple quad-
rupole mass spectrometer). An H-ESI source in positive mode was used. The parameters were as follows: the collision energy at
10.25 V for the ion pair (493.213 m/z, 475.222 m/z) and 16.62 V for the ion pair (493.213 m/z, 329.183 m/z). Peak detection was visu-
alized with the Thermo Xcalibur software.

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OPEN ACCESS Article
DL-endopeptidases production, purification

The schematic of DL-endopeptidases used in this study was showed in Figure 3B. To delineate whether the protective effect
of UC118 against colitis is NOD2 dependent, we generated the active site mutant of UC118 (W418A, C428A, H478A), named
UC118-WCH here (Figure 6A). The Sangon Biotech (Shanghai, China) was entrusted to synthesize the encoding sequences of
UC118, UC118-WCH, SagA, LAN55, LAN98, LAN50 and LAN70. The restriction enzyme sites used were: UC118, UC118-WCH,
SagA, LAN55, LAN98, LAN50 and LAN70 contained EcoR I at the 5’ end and Xho I at the 3’ end; EFOG contained BamH I at the
5’ end and Xho I at the 3’ end. All these sequences were ligated into digested pET-28a respectively and transformed
into competent E. coli BL21(DE3). During inducing protein expression, kanamycin was used at 50 mg/mL, isopropyl-b-d-thiogalacto-
side was used at 0.5 mM for UC118, UC118-WCH, LAN55, LAN98, LAN50, LAN70 and at 1 mM for SagA and EFOG. After induction of
protein expression for 8 h, bacteria were pelleted, and lysed ultrasonically. His-tagged proteins were purified using His Tagged Pro-
tein Purification Kit and desalted using phosphate buffered saline (PBS) containing 20% glycerin, followed by a clearing of potential
endotoxin by passing through a Detoxi-Gel Endotoxin Removing Gel.
Immunoblot
The anti-UC118 polyclonal antibody was generated as follows: female New Zealand white rabbits (female, 8-9 weeks, 2 kg body
weight) were immunized through repeated intradermal injections of UC118 (1:1 emulsified in Freund’s adjuvant). After the final boost,
blood was collected, and serum was prepared. The polyclonal antibody was purified using Pierce Protein A IgG Purification Kit.
Immunoblot was performed to detect whether the presence of UC118 in the lysate or culture supernatant of L. salivarius.
L. salivarius were grown in Man, Rogosa, and Sharpe medium at 37 C, 24 h without agitation. The cell lysates and culture superna-
tants were harvested, separated on sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride mem-
branes. Membranes were blocked with 5 % skim milk and incubated with rabbit polyclonal UC118 antibody (1:5000) overnight.
Expression of UC118 was detected using goat anti-rabbit IgG antibody conjugated with horseradish peroxidase and enhanced
chemiluminescence reagent kit.
Pectin/zein complex hydrogel beads

To avoid the protease attack during the oral route of administration, pectin/zein hydrogel beads were used to deliver recombinant protein
to the colon as described previously (Yan et al., 2011). Briefly, 85% alcohol containing 0.5% (wt/vol) CaCl2 was used to dissolve zein at
10 mg/ml. Proteins were mixed with 60 mg/ml pectin solution to prepare pectin/protein solution. Then, a 23 G needle was used to inhale
the pectin/protein solution into a syringe and mixed with zein solution dropwise slowly. After hardening, the beads were washed with
distilled water 4-5 times. Pectin/zein beads containing bovine serum albumin (BSA) were prepared simultaneously and used as control.
All beads sized about 2 mm with 5 mg protein on average.
Analysis of NOD2 activation

For culturomic analysis of NOD2 activation, the bacteria were harvested by centrifugation at 5000 g, 4 C for 5 min, and the pellet was
resuspended using sterile saline. Cell density was adjusted to an OD 600 nm of 1.0/mL in sterile saline and incubated aerobically or
anaerobically for 8 h. The bacteria were pelleted again and the supernatants were collected. Then, 20 mL of samples including su-
pernatant or live bacteria (at a bacterium/cells ratio of about 100 or 1000, namely log MOI = 2 or 3) were added in triplicate into
96-wells plates. Afterward, a diluted HEK Blue Detection media containing 140, 000 cells/ml HEK-Blue-NOD2 or Null cells was added
resulting in a volume of 200 mL. After incubation at 37 C in 5% CO2 for 12 h, SEAP activity was determined using a spectrophotometer
at OD635 nm. A 100 ng/ml of MDP was used as the positive control, and its DOD635 (NOD2-Null) was served as the cut-off point for
NOD2 activation.
To detect the NOD2 activation induced by the products of DL-endopeptidases-digested peptidoglycan, 100 mg of peptidoglycan
(Sigma) was predigested with mutanolysin (5 U/ml, Sigma), mixed with 20 mg of BSA or DL-endopeptidases in PBS (pH 7.5) for 16 h at
37 C. The supernatants were collected by centrifuging the mixture at 20, 000 g for 10 minutes at 4 C. Then, the peptidoglycan digests
were filtered through 5-kDa molecular weight cut-off column, the flow-through was collected and used to analyze the NOD2 activa-
tion as described above. A 100 ng/ml of MDP was used as positive control.
To detect the NOD2 activation induced by the supernatant of DL-endopeptidases-treated E. coli or S. aureus, bacteria (at a bac-
terium/cell ratio of about 1000) were centrifugated and resuspended in 1 ml sterile saline. 50 mg UC118 or SagA was added and incu-
bated for 8 h. The bacteria were pelleted by centrifuging at 20, 000 g for 10 minutes at 4 C. Then, the supernatants were collected and
filtered through 5-kDa molecular weight cut-off column, the flow-through was used to analyze the NOD2 activation as
described above.
To detect the NOD2 activation induced by fecal supernatant or intestinal homogenates, mice were gavaged with different doses
(0-5 mg/g body weight) of pectin/zein complex-coated BSA, muramidase or UC118 for 3 days. Feces and intestinal tissues were har-
vested and weighed by an analytical balance (150-200 mg). Then, 500 ml/100 mg sterile saline was added and mixed by vortexing at
max speed for 10 min. Colonic tissues were homogenized thoroughly for 10 min at 4 C. Supernatants were obtained by centrifuging
the samples at 20, 000 g for 10 minutes and filtered through a 0.22 mm pore size filters. Samples were filtered through 5-kDa molecular
weight cut-off column, the flow-through was collected and used to analyze the NOD2 activation as described above.
To quantify the NOD2 ligands levels in the human feces, fecal supernatants were prepared as described above. Standard curves
were prepared in endotoxin-free sterile saline in a concentration from 50 ng/ml to 2000 ng/ml of MDP. A volume of 20 mL of fecal

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supernatant was collected and used to analyze the NOD2 activation as described above. The standard curve of MDP was used to
calculate NOD2 ligands levels in the human feces.
Chemically induced colitis model

The standardized procedure for chemically induced colitis models was described in reference (Wirtz et al., 2017). To establish the
TNBS-induced colitis model, C57BL/6 mice (female, 6-8 weeks, 18-22 g) were anesthetized and presensitized with 150 ml 1%
TNBS (wt/vol, diluted in 4:1 mixture of acetone/olive oil) at the abdominal skin with a 1.5 3 1.5 cm area. Eight days later, mice
were intrarectally injected with 100 ml of 2.5% TNBS (wt/vol, 1:1 in ethanol) under general anesthesia and kept vertical for
0.5 min. Control mice underwent the same procedure but were administered intrarectally with 50% ethanol. Considering that loss
of Nod2 gene makes mice more prone to colitis, Nod2-/- mice were given a lower dosage of TNBS (1.5 %, wt/vol) than WT to obtain
a similar colitis severity, as described previously (Watanabe et al., 2008). For the DL-endopeptidases treatment, mice were orally
administrated with pectin/zein beads containing BSA (1 or 2 mg/g body weight) or DL-endopeptidases (1 or 2 mg/g body weight) daily
for 3 days before the TNBS challenge. Mice were assessed for body weight, fecal consistency, stool bleeding and survival every day.
At the last, mice were sacrificed, and the colons were separated and the lengths were recorded. A 2 cm segment of tissues from the
distal colon were embedded into paraffin. After cutting into 3 mm sections, Masson trichrome staining or hematoxylin-eosin (H&E)
staining was performed to evaluate collagen deposition, tissue inflammation and damage. Inflammation scores were blindly as-
sessed by independent specialists as below: no signs of inflammation scored at 0-1; one to two scattered infiltrating mononuclear
cells scored at 1-2; multiple foci of inflammatory cell infiltrating scored at 2-3; high level of inflammation, with increased vascular den-
sity and marked wall thickening scored at 3-4; maximal severity of inflammation, with transmural leukocyte infiltration and loss of
goblet cells scored at 4-5.
To establish the DSS-induced colitis model, 3% DSS solution was given to mice (female, 6-8 weeks, 18-22 g) in drinking water for
the first 5-7 days, then replaced with normal drinking water until being sacrificed. The pectin/zein beads containing proteins (2 mg/g
body weight), L. salivarius (109 CFU in 200 mL sterile saline) was orally administrated, mifamurtide (12.5 mg/g body weight) or MDP
(5 mg/g body weight) were intraperitoneally injected simultaneously with DSS treatment for 3 days. For the NOD2 inhibitor
GSK717 treatment, mice were intraperitoneally injected with 10 mg/g body weight of GSK717 one day before the DSS challenge.
Mice were assessed for body weight, fecal consistency, stool bleeding and survival every day. At the last, mice were sacrificed,
and the colons were separated, and the lengths were recorded. A 2 cm segment of tissues from the distal colon were embedded
into paraffin. After cutting into 3 mm sections, Masson trichrome staining or hematoxylin-eosin (H&E) staining was performed to eval-
uate collagen deposition, tissue inflammation and damage. Scores of inflammation-associated histological changes were evaluated
as follows: (1) Tissue damage: 0, none; 1, isolated focal epithelial damage; 2, mucosal erosions and ulcerations; 3, extensive damage
deep into the bowel wall; (2) Lamina propria inflammatory cell infiltration: 0, infrequent; 1, increased neutrophils; 2, submucosal pres-
ence of inflammatory cell clusters; 3, transmural cell infiltrations. The sum of the two sub-scores results in a combined score ranging
from 0 (no changes) to 6 (widespread cellular infiltrations and extensive tissue damage).
Intestinal permeability
The permeability of the intestinal barrier was evaluated by analyzing serum concentrations of fluorescein isothiocyanate-linked
dextran (FITC-dextran) following oral gavage as described previously (Sorribas et al., 2019). Mice were gavaged with FITC-dextran
(4 kDa) at a dose of 600 mg/kg body weight, and cardiac punctures were performed 4 h later. A fluorescence spectrophotometer was
employed to measure the serum FITC-dextran concentration with emission wavelengths at 485 nm, and excitation wavelengths
at 535 nm.
Histopathology and immuno-histochemistry

Fresh colon tissue was fixed in formaldehyde (4%) and processed under a standard histological process for sectioning and staining.
The UC118 distribution in the colon was detected. UC118 was firstly labeled with fluorescein Cy3 using Cy3 Conjugation Kit. C57BL/6
mice (female, 6-8 weeks, 18-22 g) were orally administrated with pectin/zein complex-coated Cy3-labeled UC118 or Cy3-labeled
UC118 without pectin/zein beads coated. Mice were sacrificed 6 hours later, colonic tissues were harvested and sectioned for immu-
nofluorescence analysis. Sections were blocked with normal goat serum and incubated with rabbit anti-E-cadherin (E-cadherin,
Abcam, Cat#: ab231303; RRID: AB_562059). Followed by washing and incubating with appropriate secondary antibodies and
DAPI. The distribution of UC118 in the colon was detected using fluorescence microscopy.
For the immunohistochemical staining, paraffin sections were dewaxed with xylene for 10 min, dehydrated with gradient alcohol
and rinsed in distilled water. The sections were heated in citrate buffer solution at 100 C for 40 min to retrieve antigen. Hydrogen
peroxide/methanol (30 %) was used to stop endogenous peroxidase activity (30 min at 25 C). Tissue sections were blocked with
1% BSA and incubated overnight at 4 C with the rabbit anti-PCNA antibody (Proteintech, Cat# 10205-2-AP, RRID: AB_2160330),
or rabbit anti-cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology, Cat# 9661, RRID: AB_2341188). Sections were
washed and incubated with peroxidase-conjugated anti-rabbit antibodies. After final washing, sections were visualized by 3,3=-dia-
minobenzidine with hematoxylin counterstain. Immunostaining was quantified by ImageJ software (US National Institutes of Health).
To detect apoptotic cells, a minimum of 4 fields were examined in each section, and the number of apoptotic cells/10000 cells was
determined and the apoptotic index was calculated.

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OPEN ACCESS Article
Enzyme linked immunosorbent assay (ELISA)

The pro-inflammatory cytokines (IFN-g, TNF-a, IL-17, IL-6) from serum were evaluated using ELISA kits, as described in the key
resources table, according to the manufacturer’s instructions.
Fecal microbiota transplantation

The schematic of this experiment was listed in Figure 5A. Fecal samples were divided into three groups according to the OD635
values observed from the NOD2 reporter cells assay. Fresh stools obtained from healthy control (n = 6) or CD patients (n = 12)
were dried in an oven at a temperature of 85 C for 12 h and added saline (100 mg feces with 2 mL saline). The suspensions were
centrifuged at 14, 000 rpm for 10 min, and the supernatants were ultra-filtrated using an Amicon Ultra 15 mL centrifugal filter (Milli-
pore, Massachusetts, USA) which has an exclusion limit of 5-kDa. Then 20 mL of the flow-through fraction was added into the 96-well
plates. Afterward, a diluted HEK Blue Detection media containing 140, 000 cells/ml NOD2 reports cells was added resulting in a vol-
ume of 200 mL. A 100 ng/ml of MDP was used as positive control. After incubation at 37 C in 5% CO2 for 12 h, SEAP activity was
determined using a spectrophotometer at OD 635 nm.
According to the OD635 values, fecal samples were divided into the healthy group, CD patients with high NOD2 activation (DL-
endoHigh) group and CD patients with low NOD2 activation (DL-endoLow) group. Each group have three donors, and each donor trans-
ferred to 3-4 recipient mice (Figure 5A). Fresh stool (500 mg) of donor was suspended in 5 mL of saline containing 20% glycerine and
0.5 g/L cysteines as reducing agents. After resuspension, tubes containing the feces in the reduced saline were passed through
a 100 mm cell strainer. Fecal suspensions were stored at -80 C and used for fecal transplantation. Before the fecal microbiota trans-
plantation, Nod2 knockout or its littermate control, wild-type mice (female, 6-8 weeks, 18-22 g) were treated with a cocktail of an-
tibiotics that contained 1 g/L ampicillin, 1 g/L neomycin, 0.5 g/L vancomycin, and 1 g/L metronidazole to clear their gut microbiota.
Three days washout period were ensured elimination of the antibiotics before colonization. Mice were gavaged with 100 mL of fecal
suspension daily for ten days. Then 3% DSS drinking water was added for 7 days. The colitis activity was evaluated as
described above.
To observe the long-term effects of fecal microbiota from CD patients with low NOD2 activation, donors were divided into the
Healthy, DL-endoHigh and DL-endoLow groups. The fecal suspensions from one group were pooled and gavaged to C57/BL6 (female,
6-8 weeks, 18-22 g) once every three days for 30-40 days (Figure S5A). For the DL-endopeptidase treatment, mice were gavaged
with a dose of 2 mg/g bodyweight UC118 simultaneously with fecal suspension administration. Mice were housed in specific-path-
ogen-free conditions and fed autoclaved water and food. Mice were assessed for body weight and fecal consistency every two days.
About thirty days following the fecal microbiota treatment, mice were sacrificed, the lengths of colons were recorded. A 2 cm
segment of tissues from the distal colon was embedded into paraffin. After cutting into 3 mm sections, H&E staining was performed
to evaluate tissue inflammation and damage as described above.
Quantitative real-time polymerase chain reaction

RNA was extracted from the colon tissue using RNAprep pure Tissue Kit (TIANGEN, catalog number DP431, China). The RNA was
converted to complementary DNA using the PrimeScript RT reagent Kit (TAKARA). Quantitative reverse-transcription polymerase
chain reaction of the cDNA was conducted on an ABI StepOne Plus real-time instrument (Thermo Fisher Scientific, Waltham, MA)
using the Applied Biosystems Powerup SYBR Green Master Mix and primers listed in Table S8. These primers were synthesized
by Ruibiotech (Guangzhou, China). For each gene, the housekeeping gene b-actin was used for internal normalization.
QUANTIFICATION AND STATISTICAL ANALYSIS
All the analyses in this study were done by R (Version 4.1.1). Body weight changes of mice were analyzed by two-way ANOVA. Other
datasets were analyzed using the Student’s t-test, one-way ANOVA, or non-parametric Kruskal-Wallis test, as indicated in the figure
legends. Correlation analysis was performed on Pearson Correlations. Multivariate logistic regression model was used to assess the
relationship between clinical parameters and risk for structuring/penetrating phenotypes in CD from Netherlands-IBD cohort. Two
side P value less than 0.05 was considered significant, and is represented as * P < 0.05, ** P < 0.01, ** P < 0.001. The number of re-
peats for each experiment and detailed descriptions of statistical tests were specified in the respective figure legends.
ADDITIONAL RESOURCES
Samples in humans were performed under an approved protocol registered under ClinicalTrials: NCT04924686.

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Article

Gut microbial DL-endopeptidase alleviates Crohn’s

d DL-endopeptidase is depleted in CD patients and negatively

d Fecal microbiota of CD patients with low DL-endopeptidase

d Supplementation with DL-endopeptidase protects mice

Gao et al., 2022, Cell Host & Microbe 30, 1–15

Medical University, Guangzhou, Guangdong 510515, China

*Correspondence: r.k.weersma@mdl.umcg.nl (R.K.W.), hxl2027315@smu.edu.cn (X.H.), biodegradation@gmail.com (H.Z.)

INTRODUCTION Crohn’s disease (CD), Blau syndrome, asthma, arthritis, and

2 Cell Host & Microbe 30, 1–15, October 12, 2022

Cell Host & Microbe 30, 1–15, October 12, 2022 3

Figure 3. DL-endopeptidase determines the generation of NOD2 ligands in the gut

4 Cell Host & Microbe 30, 1–15, October 12, 2022

(legend continued on next page)

6 Cell Host & Microbe 30, 1–15, October 12, 2022

Cell Host & Microbe 30, 1–15, October 12, 2022 7

(legend continued on next page)

Cell Host & Microbe 30, 1–15, October 12, 2022 9

10 Cell Host & Microbe 30, 1–15, October 12, 2022

Cell Host & Microbe 30, 1–15, October 12, 2022 11

d KEY RESOURCES TABLE DECLARATION OF INTERESTS

12 Cell Host & Microbe 30, 1–15, October 12, 2022

Cell Host & Microbe 30, 1–15, October 12, 2022 13

14 Cell Host & Microbe 30, 1–15, October 12, 2022

Cell Host & Microbe 30, 1–15, October 12, 2022 15

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

e1 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022 e2

e3 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

Data and code availability

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Patients and samples preparation

Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022 e4

Cell lines and culture conditions

Bacterial strains and culture conditions

Sequence search and protein bioinformatic analysis

Profiling PGH-encoding genes in 1520 gut reference genomes

Public metagenomic datasets and quantification of PGH-encoding genes

Comparative genomic analysis

e5 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

Fecal 16S rRNA microbial analysis

Metagenomic sequence preprocessing

Public transcriptional data and analysis

NOD2 polymorphisms testing

Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022 e6

DL-endopeptidases production, purification

Pectin/zein complex hydrogel beads

Analysis of NOD2 activation

e7 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

Chemically induced colitis model

Histopathology and immuno-histochemistry

Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022 e8

Enzyme linked immunosorbent assay (ELISA)

Fecal microbiota transplantation

Quantitative real-time polymerase chain reaction

QUANTIFICATION AND STATISTICAL ANALYSIS

e9 Cell Host & Microbe 30, 1–15.e1–e9, October 12, 2022

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