# The l,d-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile

Ana M Oliveira Paiva, Pascal Courtin, Glenn Charpentier, Imane

Oueled-Chama, Olga Soutourina, Marie-Pierre Chapot-Chartier, Johann

Peltier

► To cite this version:

Ana M Oliveira Paiva, Pascal Courtin, Glenn Charpentier, Imane Oueled-Chama, Olga Soutourina, et al.. The l,d-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile. iScience, 2025, 28, pp.112227. 10.1016/j.isci.2025.112227. hal-05187111

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# The L,D-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Ana M. Oliveira Paiva, Pascal Courtin, Glenn Charpentier, Imane Oueled-Chama, Olga Soutourina, Marie-Pierre Chapot-Chartier, Johann Peltier

## Correspondence

johann.peltier@i2bc.paris-saclay.fr

## In brief

Biochemistry; Microbiology; Pharmacology

## Highlights

* C. difficile relies on non-classical L,D-transpeptidases for peptidoglycan cross-linking

* The spore cortex of C. difficile is exclusively cross-linked by L,D-transpeptidation

* D,D-carboxypeptidases are the primary target of β-lactams in C. difficile

* D,D-carboxypeptidases controls the mode of transpeptidation in C. difficile

<figure><img src="image_3.png" /></figure>
## Article

# The L,D-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in *Clostridioides difficile*

Ana M. Oliveira Paiva,<fnref n="1" /> Pascal Courtin,<fnref n="2" /> Glenn Charpentier,<fnref n="1" /> Imane Oueled-Chama,<fnref n="1" /> Olga Soutourina,<fnref n="1" /> Marie-Pierre Chapot-Chartier,<fnref n="2" /> and Johann Peltier<fnref n="1" />,<fnref n="3" />,*

## SUMMARY

The resistance of *Clostridioides difficile* to the β-lactam antibiotics cephalosporins, which target the peptido-glycan (PG) assembly, is a leading contributor to the development of *C. difficile* infections. *C. difficile* has an original PG structure with a predominance of 3→3 cross-links generated by L,D-transpeptidases (LDTs). *C. difficile* forms spores and we show that the spore cortex PG contains exclusively 3→3 cross-links. PG and spore cortex of *C. difficile* cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTs. The D,D-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the L,D-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in *C. difficile*.

## INTRODUCTION

*Clostridioides difficile* is the primary cause of antibiotic-associated nosocomial diarrhea in adults.<fnref n="1" /> *C. difficile* forms spores that are essential for the dissemination of *C. difficile* infections (CDI).<fnref n="2" /> Antibiotic exposure resulting in gastrointestinal dysbiosis is commonly associated with the development of CDI.<fnref n="1" /> β-lactam antibiotics, particularly cephalosporins, are widely recognized as key factors in the development of CDI. However, the mechanism underlying the intrinsic resistance of *C. difficile* to these antibiotics remains poorly understood.<fnref n="3" />,⁴ β-lactams target the cell wall peptidoglycan (PG) assembly. PG is a polymeric macromolecule consisting of linear glycan chains cross-linked by short peptides.⁵ The D,D-transpeptidase (DDT) activity of penicillin-binding proteins (PBPs) connects the fourth amino acid of one peptide side chain to the third amino acid of another, forming 4→3 cross-links (Figure 1A). However, 3→3 cross-links generated by L,D-transpeptidases (LDTs) presenting a YkuD-like domain have also been reported (Figure 1B).⁶ In addition to their cross-linking activity, both DDTs and LDTs catalyze exchange reactions where the terminal D-Ala in pentapeptides and tetrapeptides, respectively, are replaced by a non-canonical D-amino acid (NCDAA).⁷,⁸ DDTs, which are the primary targets of β-lactam antibiotics, use native PG precursors containing a penta-peptide stem as acyl donors for cross-linking. In contrast, LDT activity requires a tetrapeptide stem as the acyl donor substrate.⁶ This substrate is typically produced by D,D-carboxypep-

tidases that cleave the fifth residue from the pentapeptide chain (Figure 1B).⁹⁻¹¹ LDTs have the potential to cf. resistance to β-lactam antibiotics, as they are solely inhibited by β-lactam antibiotics of the carbapenem class.¹²

*C. difficile* is the only known Bacillota with predominant 3→3 PG cross-links.⁶,¹³,¹⁴ Recent studies revealed that a mutant strain of *C. difficile* R20291, which lacks all three LDT paralogues, is not impaired in the production of 3→3 cross-links.¹⁵,¹⁶ Furthermore, two new enzymes with a VanW catalytic domain have been identified as the missing LDTs.¹⁶ However, the role of 3→3 cross-linking in mediating cephalosporin resistance in *C. difficile* remains unclear. While the structure of the *C. difficile* spore cortex, a modified layer of PG, has also been characterized, the type of cross-links present has not been defined.¹⁷

In this study, we showed that the spore cortex PG of *C. difficile* only contains 3→3 cross-links. As for the PG of vegetative cells, 3→3 cross-links were still present in the spore cortex of a *C. difficile* strain lacking the three YkuD domain-containing LDTs, suggesting the implication of the VanW domain proteins. By treating *C. difficile* cells with subinhibitory concentrations of β-lactams, including cephalosporins and carbapenems, we discovered that at least one D,D-carboxypeptidase, which is necessary for the production of LDT substrates, is the primary target of these antibiotics and is differentially sensitive to them. In contrast, PBPs generating the 4→3 cross-links are not inhibited by these concentrations of antibiotics. Thus, cephalosporin

<figure><img src="image_4.png" /></figure>

<footnote n="1">¹Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France</footnote>

<footnote n="2">²Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, Jouy-en-Josas, France</footnote>

<footnote n="3">³Lead contact</footnote>

<footnote>*Correspondence: johann.peltier@i2bc.paris-saclay.fr</footnote>

<footnote>https://doi.org/10.1016/j.isci.2025.112227</footnote>
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /><figcaption>**Figure 1.** Formation of PG cross-links by the D,D- and the L,D-transpeptidation pathway</figcaption></figure>

(A) The D,D-transpeptidase (DDT) activity of the PBPs cleaves the terminal D-Ala of a native pentapeptide stem donor and subsequently generates a 4→3 cross-link with an acceptor stem.

(B) L,D-transpeptidases (LDT) cleave the terminal D-Ala of a tetrapeptide stem donor and subsequently generate a 3→3 cross-link with an acceptor stem. Enzymes with D,D-carboxypeptidase (D,D-CPase) activity produce the essential tetrapeptide donors from pentapeptide native stems. G, N-acetylglucosamine; M, N-acetylmuramic acid.

resistance in *C. difficile* is not dependent on PG cross-linking by LDTs and instead relies on the poor inhibition of PBPs by this anti-biotic family. These findings suggest that the growth arrest induced by β-lactams is mediated by the inhibition of the L,D-transpeptidation pathway in *C. difficile*.

## RESULTS

### A strain lacking the three YkuD proteins still predominantly produces 3→3 cross-links

To evaluate the role of the three proteins harboring the catalytic domain YkuD on the PG structure of *C. difficile* 630Δ*erm*, we generated a triple mutant of the corresponding genes *ldt*<sub>Cd1</sub> (*CD2963*), *ldt*<sub>Cd2</sub> (*CD2713*), and *ldt*<sub>Cd3</sub> (*CD3007*) (*Figures S1A* and S1B).¹⁴ This mutant, hereby referred as ΔΔΔ*ldt*, was confirmed via whole-genome sequencing and no additional mutation could be detected (see STAR Methods). The growth rate of the ΔΔΔ*ldt* strain was similar to that of the parental strain 630Δ*erm* (*Figure S1C*).

Wild-type and ΔΔΔ*ldt* cells were collected in exponential growth and the purified PG was digested with mutanolysin to generate muropeptides. Muropeptides were analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) (*Figure 2A*) and their structure was deduced from their *m/z* values and fragmentation patterns obtained by MS/MS. The structure of all identified muropeptides is summarized in Table S1. The muropeptide composition of the ΔΔΔ*ldt* strain was largely similar to that of the parental strain (*Figure 2A*; Table S1). The main difference was a strong reduction of the abundance of muropeptides containing a tetrapeptide stem ending in Gly (peaks 4 and 11) in the mutant compared to the wild-type strain (*Figures 2A and 2B; Table S1*), revealing a role of at least one of the three enzymes in the exchange reaction of the terminal D-Ala in tetrapeptide stems

with Gly. Additionally, a small but significant decrease of the dimers cross-linked by L,D-transpeptidation, which results in a reduction of the cross-linking index was observed in the ΔΔΔ*ldt* strain compared to the parental strain (*Figures 2C and 2D*). Nonetheless, 3→3 cross-links remained predominant in the triple mutant strain, indicating the presence of LDTs with a different catalytic domain compensating for the loss of the canonical LDTs.

To confirm the impact of the mutations on the exchange reaction, *C. difficile* cells were labeled with the amino acid probe HCC-amino D-alanine (HADA) during exponential growth,¹⁸,¹⁹ and visualized using fluorescence microscopy (*Figures S2A and S2B*). The cell length and width between the triple mutant and the parental strain were identical (*Figures S2C and S2D*). In pre-divisional cells, the intensity of the HADA staining significantly increased at mid-cell, as expected for the septum formation (*Figures S2B, S2E, and S2F*). In dividing cells, a gradient of fluorescence was observed with a concentrated signal at one of the poles, which presumably corresponds to the newly formed pole, and a fading signal at the other pole (*Figures S2B and S2F*). The triple mutant showed a similar fluorescent labeling profile to the wild type around the cells, but an overall significant decrease in fluorescence intensity (*Figures S2B and S2E*). This result suggests that HADA incorporation through the exchange reaction into the PG of *C. difficile* is partially dependent on the activity of at least one of the LDTs.

### *Ldt*<sub>Cd1</sub> contributes to the exchange reaction and the generation of 3→3 cross-links

To elucidate the contribution of the three LDTs to exchange reactions and 3→3 cross-linking, the muropeptide profile of the different single and double mutants was also analyzed (*Figure S3; Table S1*). The abundance of tetrapeptides ending in Gly and of 3→3 cross-links in the strains deleted of *ldt*<sub>Cd2</sub> and/or *ldt*<sub>Cd3</sub>
<figure><img src="image_7.png" /><figcaption>Figure 2. Impact of the *ldt* single, double and triple deletions on the PG structure of *C. difficile* vegetative cells</figcaption></figure>

(A) LC-MS chromatograms (total ion current [TIC]) of muropeptides from vegetative cells of *C. difficile* 630Δ*term* wild-type (WT) and ΔΔΔ*ldt* strains. Major peaks are labeled with numbers referring to Table S1. See also Table S1 for the structure of all identified muropeptides. Data are representative of three independent experiments.

(B) Abundance of muropeptides containing a modified tetrapeptide stem with the LDT-mediated exchange of the terminal D-Ala by a Gly in the PG from *C. difficile* WT and *ldt* mutant strains.

(C) Abundance of muropeptide dimers with a 3→3 cross-link relative to the total cross-links in dimers in the PG from *C. difficile* WT and *ldt* mutant strains.

(D) Cross-linking index of PG from *C. difficile* WT and *ldt* mutant strains.

All graphs represent mean ± SEM and include individual data points; *n* = 3 independent experiments for WT and ΔΔΔ*ldt* and *n* = 2 independent experiments for the other mutant strains. \*p* ≤ 0.05 and \*\*\*p* ≤ 0.0001 by a one-way ANOVA followed by a Dunnett's multiple comparisons test.
A

<figure><img src="image_8.png" /></figure>

WT

ΔΔΔldt

B

<figure><img src="image_9.png" /></figure>

C

<figure><img src="image_10.png" /></figure>

WT

ΔΔΔldt

<caption>Figure 3. Impact of the *ldt* triple deletion on the PG structure of *C. difficile* spore cortex</caption>

(A) LC-MS chromatograms (TIC) of muropeptides from the spore cortex of *C. difficile* 630Δ*erm* wild-type (WT) and ΔΔΔ*ldt* strains. Major peaks are labeled with numbers referring to Table S2. See also Table S2 for the structure of all identified muropeptides. Data are representative of three independent experiments.

(B) Abundance of NAG deacetylation in the spore cortex from *C. difficile* WT and ΔΔΔ*ldt* strains.

(C) Abundance of MAL and NAM in the spore cortex from *C. difficile* WT and ΔΔΔ*ldt* strains. Percentages of unsubstituted NAM (no peptide) and of NAM substituted with a dipeptide, a tripeptide or a tetrapeptide, or involved in cross-linking are represented.

All graphs represent mean ± SEM and include individual data points; *n* = 3 independent experiments.

were similar to those of wild type (Figures 2B and 2C). However, deletion of either *ldt*<sub>Cd1</sub>, *ldt*<sub>Cd1</sub>, and *ldt*<sub>Cd2</sub> or *ldt*<sub>Cd1</sub> and *ldt*<sub>Cd3</sub> recapitulated the decrease of tetrapeptides ending in Gly observed in the ΔΔΔ*ldt* strain. The proportion of 3→3 cross-links was also reduced in the Δ*ldt*<sub>Cd1</sub>Δ*ldt*<sub>Cd2</sub> and Δ*ldt*<sub>Cd1</sub>Δ*ldt*<sub>Cd3</sub> double mutants with an impact on the cross-linking index (Figures 2C and 2D). Thus, these data indicate that the PG modifications observed in the triple mutant strain are mostly mediated by the *ldt*<sub>Cd1</sub> deletion.

## The spore cortex of *C. difficile* is cross-linked by L,D-transpeptidation

To determine the mode of cross-linking in the spore cortex, spores of *C. difficile* 630 Δ*erm* wild type were purified and their PG cortex was isolated. Muropeptides were generated using mutanolysin, analyzed by UHPLC-MS/MS (Figure 3A), and their structure was deduced from their *m/z* values and fragmentation patterns obtained by MS/MS. All the identified peaks are sum-

marized in Table S2. In agreement with a previous report,¹⁷ 39.5% ± 3.7% of the NAG residues were found to be N-deacetylated in the spore cortex of *C. difficile* wild type (Figure 3B). In addition, 31.1 ± 2.9% of the NAM residues were converted to the spore-specific MAL in *C. difficile* spore cortex (Figure 3C). This modification prevents the cleavage between the NAM and NAG residues by mutanolysin, leading to the presence of oligosaccharides among the generated muropeptides. Most of the muropeptides still containing an unmodified NAM residue lacked any stem peptide (34.7 ± 3.1%) or harbored a tetrapeptide stem (30.6 ± 4.6%) (Figure 3C).

Among the identified muropeptides, only two corresponded to dimers and no trimer was detected (Table S2). Thus, the spore cortex of *C. difficile* is very weakly cross-linked with a cross-linking index of 1.5 ± 0.3% (Figure 3C). The two dimers had the same structure with a tripeptide and a tetrapeptide stem and both contained a 3→3 cross-link as revealed by the loss of one C-terminal alanine (mass loss of 89.048 Da) from the acceptor
<figure><img src="image_11.png" /><figcaption>Figure 4. MS/MS analysis of the main muropeptide dimer from the spore cortex of C. difficile wild type</figcaption></figure>

(A) MS/MS analysis of the molecular ion [M+2H]²⁺ (m/z = 855.887 with z = 2) detected in peak 14 with a retention time of 7.99 min in Figure 4A and Table S2. Fragments (squared) were detected as [M + H]⁺ (z = 1) or [M+2H]²⁺ (z = 2) adducts. Fragments resulting from loss of a water molecule are not squared. The fragments allowing assignment of the 3-3 cross-links are highlighted in yellow.

(B) Structures inferred from the MS/MS analysis are presented. The loss of one alanine from the C-terminal end of different ions (mass loss of 89.048) establishes the presence of a 3→3 cross-link.

tetrapeptide stem in their MS-MS fragmentation spectrum (Figures 4 and S4). Thus, the spore cortex of *C. difficile* contains only 3→3 cross-links.

Next, we analyzed the spore PG structure of the ΔΔΔ/dt strain to assess the implication of the LDTs in the formation of the 3→3

cross-links. However, the muropeptide profile of the mutant was identical to that of the wild-type strain and no decrease of the 3→3 cross-links was observed (Figure 3; Table S2). These data indicate that either the canonical LDTs do not contribute to the cross-linking of the spore cortex or their loss is compensated by
<figure><img src="image_12.png" /><figcaption>Figure 5. Impact of different β-lactam antibiotics on the PG structure of vegetative cells of C. difficile wild type and ΔΔΔIdt</figcaption></figure>

(A) LC-MS chromatogram of muropeptides from vegetative cells of C. difficile wild-type (WT) strain grown in the presence of subinhibitory concentrations of cefoxitin. Peak labels refer to Table S1. New major peaks observed in the presence of the antibiotics are labeled with letters. See also Table S1 for the structure of all identified muropeptides. Data are representative of three independent experiments.

(B) Abundance of muropeptides with a tetrapeptide or a pentapeptide stem (free side or acceptor chain in multimers) in the PG of C. difficile wild-type and ΔΔΔIdt strains grown in the absence of antibiotics (no ATB) or in the presence of cefoxitin or meropenem.

(C) Abundance of muropeptide dimers with a 4→3 and a 3→3 cross-link relative to total muropeptides in the PG of C. difficile wild-type and ΔΔΔIdt strains grown in the absence of antibiotics (no ATB) or in the presence of cefoxitin or meropenem.

(legend continued on next page)
the activity of at least one of the two novel LDTs with a VanW
domain.¹⁶

The YkuD domain proteins do not contribute to β-lactam
resistance in C. difficile

We investigated the effect of the triple *ldt* deletion on β-lactam
resistance in *C. difficile*. Loss of the three L,D-transpeptidases
did not alter the minimum inhibitory concentration (MIC) of
different members of the β-lactam antibiotics class (Table S3).
In addition, growth of the ΔΔΔ*ldt* and the parental strains in
the presence of subinhibitory concentrations of different β-lac-
tams (1/2 MIC) were similar (Figure S5). We reasoned that the
gene expression of the VanW domain LDTs or other enzymes
of the PG polymerization machinery might be induced in the
presence of β-lactams to compensate for the loss of the three
YkuD proteins. To test this hypothesis, 630Δ*erm* and ΔΔΔ*ldt*
were grown in the presence of subinhibitory concentrations of
the cephalosporin antibiotic ceftriaxone (1/2 MIC) and the sam-
ples were subjected to RNA sequencing. Whereas genes
involved in stress response, such as type I toxin-antitoxin sys-
tems were induced in ΔΔΔ*ldt*, no gene related to PG meta-
bolism, including the VanW proteins (CD1436 and CD2149 in
630Δ*erm*) or encoding PG-associated proteins could be identi-
fied as differently expressed (Table S4). These data indicate that
the YkuD domain proteins are dispensable for β-lactam resis-
tance in *C. difficile* 630Δ*erm*.

The L,D-transpeptidation pathway is inhibited by
subinhibitory concentrations of cephalosporin and
carbapenem antibiotics in *C. difficile*

To examine the impact of β-lactams on PG cross-linking, the
PG structure of vegetative cells of the wild-type strain grown
in the presence of 1/2 MIC of the cephalosporin cefoxitin was
analyzed. *C. difficile* exposure to this antibiotic led to drastic
PG structure modifications, in comparison to the non-treated
strain, with the appearance of new abundant muropeptides
with a pentapeptide stem to the detriment of tetrapeptide
stems (Figures 2A, 5A, and 5B; Table S1). Muropeptides with
a pentapeptide stem accounted for 30 ± 1.9% of the total mur-
opeptides in the cefoxitin-treated strain when they represented
only 1 ± 0.1% in the untreated strain. Conversely, the abun-
dance of tetrapeptides decreased from 66.9 ± 0.5% to 36.1 ±
2.5% in the presence of the antibiotic (Figures 5A and 5B;
Table S1). These changes were accompanied by a strong
decrease of the abundance of muropeptide dimers containing
3→3 cross-links (Figure 5C; Table S1). Remarkably, the
cross-linking index remained similar in the absence and in the
presence of antibiotics as the 3→3 cross-link reduction was
compensated by an equivalent increase of the 4→3 cross-

linked dimers (Figures 5C and 5D). Overall, the proportion of
3→3 cross-links relative to the total cross-links shifted from
76.3 ± 0.7% in the non-treated wild-type strain to 40.3 ±
2.9% in the strain treated with cefoxitin (Figure 5E). Similar re-
sults were obtained when analyzing the PG structure of the
ΔΔΔ*ldt* strain grown in the presence of cefoxitin, revealing
the involvement of the non-canonical LDTs in the synthesis of
the residual 3→3 cross-links (Figures 5B–5E and S6A;
Table S1). Moreover, treatment of the ΔΔΔ*ldt* strain with ceftri-
axone, another antibiotic of the cephalosporin class, resulted
in similar PG modifications, although at a lesser extent (Figures
S7A–S7E). Thus, these data strongly suggest that cephalospo-
rins inactivate at high concentrations one or several D,D-car-
boxypeptidase(s), whose activity is required to produce the
substrate of the LDT. They also reveal that at least one PBP
catalyzing the formation of the 4→3 cross-linking reaction is
not inactivated by these antibiotics.

C. difficile 630Δerm is resistant to most cephalosporins but susceptible to carbapenems (Table S3).²⁰ To assess whether carbapenems also impacted the activity of D,D-carboxypeptidases in C. difficile, PG structure of wild-type and ΔΔΔldt cells treated with 1/2 MIC of meropenem was analyzed (Figures 5B, S6A, and S6B; Table S1). As with cephalosporins, meropenem induced the appearance of several major muropeptide peaks with pentapeptide stems and modified the balance between 4→3 and 3→3 cross-links to the benefits of 4→3 cross-links in the two strains (Figures 5B–5E). Treatment of the ΔΔΔldt strain with imipenem, another antibiotic of the carbapenem class led to the same PG modifications (Figures S7A–S7E).

Another remarkable effect of the antibiotic treatments on the
PG structure was the presence of a high proportion of muropep-
tides with a pentapeptide stem ending with a non-canonical
amino acid (NCDAA: Gly, Phe, Leu, or Val) (Table S1; Figures
5F and S7F). The incorporation of NCDAA into the PG was shown
to regulate the cell wall structure in response to environmental
stresses in Vibrio cholerae and the same mechanism was sug-
gested in Bacillus subtilis.⁸,²¹

Altogether, these data strongly suggest that at least one D,D-
carboxypeptidase plays a pivotal role in the control of the
mode of transpeptidation in C. difficile. This D,D-carboxypepti-
dase is the primary target of antibiotics of the β-lactam family
and is differentially susceptible to these antibiotics with an effi-
cient inhibition by low concentrations of carbapenems but only
by high concentrations of cephalosporins. The associated
reduction of 3→3 cross-linking, resulting from the limited LDT
substrate availability, highlights that cephalosporin resistance
is not primarily mediated by LDTs in C. difficile but rather relies
on PBPs resistant to this class of antibiotics.

(D) Cross-linking index of the PG of *C. difficile* wild-type and ΔΔΔ*ldt* strains grown in the absence of antibiotics (no ATB) or in the presence of cefoxitin or meropenem.

(E) Abundance of muropeptide dimers with a 3→3 cross-link relative to the total cross-links in dimers in the PG of *C. difficile* wild-type and ΔΔΔ*ldt* strains grown in the absence of antibiotics (no ATB) or in the presence of cefoxitin or meropenem.

(F) Abundance of muropeptides with a pentapeptide stem ending with a non-canonical d-amino-acid (NCDAA: Gly, Phe, Leu, or Val) (Penta-NCDAA⁵) relative to the total pentapeptide muropeptides in the *C. difficile* wild-type and ΔΔΔ*ldt* strains grown in the presence of cefoxitin or meropenem.

All graphs represent mean ± SEM and include individual data points; n = 3 independent experiments. ***p* ≤ 0.01 and ****p* ≤ 0.001 by a two-way ANOVA followed by a Dunnett's multiple comparisons test.
DISCUSSION

In agreement with recent studies,¹⁵,¹⁶ we showed here that a *C. difficile* 630Δ*erm* strain lacking the three predicted LDTs presented only a slight reduction of 3→3 cross-links and a decrease in the exchange reaction. The two novel LDTs with a VanW domain most likely compensate for the loss of the other enzymes as recently shown in the strain R20291.¹⁶ PG analysis of the different *Ldt* single and double mutant strains further identified Ldt<sub>Cd1</sub> as the enzyme mediating most of the observed changes. This result is contrasting with *in vitro* assays of the three recombinant LDTs showing that Ldt<sub>Cd1</sub> is poorly active and has a weak activity in catalysis of the exchange reaction in comparison to Ldt<sub>Cd2</sub> and Ldt<sub>Cd3</sub>.¹⁵,²²

We showed the abundant presence of muramic-δ-lactams and N-acetylmuramic acids with no peptide stem, which results in a low amount of cross-links, in the spore cortex of *C. difficile*. Furthermore, we discovered that 3→3 cross-links are largely predominant, if not exclusive, in the spore cortex. In *B. subtilis*, the enzymes required for the cross-linking of the spore cortex are produced in the mother cell and their expression is under control of SigE,²³ a sporulation-specific sigma factor. Previous transcriptomic analyses identified *LdtCd3* gene as a member of the SigE regulon in *C. difficile*.²⁴⁻²⁶ In addition, LdtCd3 was shown to display a strong LDT activity *in vitro*.¹⁵,²² Yet, deletion of the three LDT-encoding genes had no impact on the abundance of the cross-links in the cortex PG, implying the involvement of the non-canonical LDTs. Future work will be required to identify which LDTs contribute to the formation of the spore cortex PG.

Among the inhibitors of PG polymerization, β-lactam antibiotics stand out as the most clinically significant. The β-lactam ring found in all β-lactam antibiotics interacts with the essential nucleophilic serine of PBPs, which can display DDT, endopeptidase, or D,D-carboxypeptidase activity.²⁷ This interaction results in the creation of a stable acyl-enzyme adduct that interferes with catalysis. In contrast, LDTs are slowly acylated by β-lactams of the penicillin and cephalosporin classes and the resulting acyl-enzymes are rapidly hydrolyzed.²⁸ Thus, LDTs are insensitive to these antibiotics but are still efficiently inactivated by carbapenems.²⁹,³⁰ In agreement, activation of the L,D-transpeptidation pathway in *Enterococcus faecium* and *Escherichia coli* conferred broad-spectrum resistance to β-lactams, with the exception of carbapenems.⁹,²⁹ Conversely, we showed that this pathway is inhibited by both cephalosporins and carbapenems in *C. difficile*, revealing that it does not mediate resistance to these drugs. An inhibition of the DDT activity of PBPs could explain the accumulation of muropeptides with a pentapeptide stem observed in the presence of the antibiotics. However, this hypothesis is not supported by our data as 4→3 PG cross-links were more abundant in antibiotic-treated cells. In line with this result, biochemical analyses of the purified class A PBP1 and the class B PBP2 of *C. difficile*, which are respectively essential for cross-links synthesis during vegetative cell division and elongation, revealed that both enzymes are insensitive to most cephalosporins and carbapenems.³¹,³² The only other explanation to the observed structural changes is the inhibition of at least one D,D-carboxypeptidase. Activation of the L,D-transpeptidation pathway leading to β-lactam resistance in *E. faecium* and

*E. coli* requires the production of a β-lactam-insensitive D,D-carboxypeptidase to generate the tetrapeptide substrate of LDTs.⁹,¹¹ In contrast, production of the essential LDT-tetrapeptide substrate by a β-lactam-sensitive D,D-carboxypeptidase in *C. difficile* results in the suppression of LDT activity in the presence of the antibiotics. Altogether, our data support the hypothesis that at least one D,D-carboxypeptidase, controlling substrate availability for LDTs, is the primary target for cephalosporins and carbapenems in *C. difficile*. Since 3→3 cross-links are essential for viability in this bacterium,¹⁶ substrate deprivation for LDTs most likely represents the original mechanism by which cephalosporins and carbapenems impact *C. difficile* survival. *C. difficile* 630 encodes 4 putative β-lactam-sensitive D,D-carboxypeptidase belonging to the PBP family (pfam00768) with two of them being required for formation of heat-resistant spores.³³ Future research will seek to identify which D,D-carboxypeptidase is involved, as this enzyme represents an attractive target for the development of new therapies to fight *C. difficile* infections.

Limitations of the study

Despite its significant implications, our study has several limitations. First, this investigation was conducted using only the laboratory strain 630Δ*erm* of *C. difficile* as a model. While the primary findings of this study are likely applicable to clinical strains of *C. difficile*, this remains to be experimentally demonstrated. Second, our study strongly suggests that an enzyme with a D,D-carboxypeptidase activity and belonging to the PBP family is the killing target of β-lactams in *C. difficile*. However, further research is required to explore the impact of a broader range of β-lactam antibiotics on the PG structure and identify the gene encoding this D,D-carboxypeptidase. Lastly, subsequent studies are needed to investigate the reasons for the essentiality of the L,D-transpeptidation pathway in *C. difficile*.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Johann Peltier (johann.peltier@i2bc.paris-saclay.fr).

Materials availability

Strains generated in this study are available from the [lead contact](mailto:lead contact) without restrictions.

Data and code availability

* LC-MS/MS datasets have been deposited in the GLYCOPOST repository: GPST000426. Accession number is listed in the *key resources table*. RNA-seq and whole genome sequencing data have been deposited to the NCBI Sequence Read Archive (SRA). Accession number is listed in the *key resources table*.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the [lead contact](mailto:lead contact) upon request.

ACKNOWLEDGMENTS

We are grateful to Alain Guillot (Micalis, INRAE) and François Fenaille (CEA, Université Paris-Saclay) for helpful discussions. We acknowledge ChemSyBio team (Micalis, INRAE, Jouy-en-Josas) for access to LC-MS/MS facilities. The present work has benefited from Imagerie-Gif core facility supported by the
Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBiolmaging, and ANR-11-IDEX-0003-02/ Saclay Plant Sciences). This work was funded by the Agence Nationale de la Recherche (ANR-20-CE15-0003-DIFFICROSS to J.P.).

## AUTHOR CONTRIBUTIONS

Conceptualization, A.M.O.P., M.-P.C.-C., and J.P.; methodology, A.M.O.P. and J.P.; investigation, A.M.O.P., G.C., P.C., and I.O.-C.; writing – original draft, A.M.O.P. and J.P.; writing – review & editing, A.M.O.P., O.S., M.-P.C.-C., and J.P.; visualization, A.M.O.P., P.C., M.-P.C.-C., and J.P.; supervision, A.M.O.P. and J.P.; funding acquisition, J.P.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODELS AND STUDY PARTICIPANT DETAILS

* Bacterial strains and growth conditions

* METHOD DETAILS

* Construction of the C. difficile Idt mutants

* Whole genome sequencing

* Fluorescence microscopy

* Spore purification

* Purification and structural analysis of PG

* Minimum inhibitory concentration (MIC) determination

* RNA sequencing

* QUANTIFICATION AND STATISTICAL ANALYSES

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2025.112227.

Received: June 24, 2024

Revised: February 6, 2025

Accepted: March 12, 2025

Published: March 16, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Bacterial strains</strong></td></tr><tr><td>E. coli NEB10β</td><td>New England Biolabs</td><td>C3019H</td></tr><tr><td>E. coli HB101(RP4)</td><td>Laboratory stock</td><td>N/A</td></tr><tr><td>C. difficile 630Δerm</td><td>Laboratory stock, Hussain et al.<sup>34</sup></td><td>N/A</td></tr><tr><td>C. difficile CD572: 630Δerm<br>ΔCD2713 ΔCD2963 ΔCD3007 (ΔΔΔldt)</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD475: 630Δerm ΔCD2963 (ldt<sub>CD1</sub>)</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD544: 630Δerm ΔCD3007 (ldt<sub>CD3</sub>)</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD545 630Δerm ΔCD2713 (ldt<sub>CD2</sub>)</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD565 630Δerm ΔCD2713 ΔCD2963</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD561 630Δerm ΔCD2713 ΔCD3007</td><td>This paper</td><td>N/A</td></tr><tr><td>C. difficile CD547 630Δerm ΔCD2963 ΔCD3007</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Brain heart infusion broth</td><td>BD</td><td>237500</td></tr><tr><td>Bacto peptone</td><td>BD</td><td>211677</td></tr><tr><td>Protéose peptone n° 2</td><td>Gibco</td><td>212120</td></tr><tr><td>Tris-base</td><td>Merck</td><td>T6687</td></tr><tr><td>Ammonium sulfate</td><td>Merck</td><td>A4418</td></tr><tr><td>LB broth</td><td>Merck</td><td>L3022</td></tr><tr><td>Chloramphenicol</td><td>Merck</td><td>C0378</td></tr><tr><td>Ampicillin</td><td>Merck</td><td>A9518</td></tr><tr><td>Thiamphenicol</td><td>Merck</td><td>T0261</td></tr><tr><td>NEBuilder HiFi DNA Assembly</td><td>New England Biolabs</td><td>E2621X</td></tr><tr><td>Oxoid Clostridioides difficile Selective supplement</td><td>Thermo Scientific</td><td>SR0096E</td></tr><tr><td>NucleoSpin Micobial DNA Mini Kit</td><td>Macherey-Nagel</td><td>740235.50</td></tr><tr><td>HADA</td><td>Tocris Bioscience</td><td>6647</td></tr><tr><td>HistoDenz</td><td>Merck</td><td>D2158</td></tr><tr><td>Cefoxitin sodium salt</td><td>Thermo Scientific</td><td>455280050</td></tr><tr><td>Ceftriaxone sodium salt</td><td>Thermo Scientific</td><td>455420050</td></tr><tr><td>Meropenem</td><td>Thermo Scientific</td><td>462810010</td></tr><tr><td>Imipenem</td><td>Thermo Scientific</td><td>455322500</td></tr><tr><td>Amoxicillin</td><td>Merck</td><td>A8523</td></tr><tr><td>Oxacillin sodium salt</td><td>Thermo Scientific</td><td>455440010</td></tr><tr><td>Urea</td><td>Thermo Scientific</td><td>A12360.36</td></tr><tr><td>Tris-HCl</td><td>Euromedex</td><td>EU0011-C</td></tr><tr><td>Sodium dodecyl sulfate 20%</td><td>Euromedex</td><td>EU0460-B</td></tr><tr><td>DTT</td><td>Euromedex</td><td>EU0006-B</td></tr><tr><td>Desoxyribonuclease I (DNase I)</td><td>Merck</td><td>D4527</td></tr><tr><td>Ribonuclease A (RNase)</td><td>Merck</td><td>R5503</td></tr><tr><td>Lipase</td><td>Merck</td><td>62301</td></tr><tr><td>Pronase</td><td>Roche</td><td>10165921001</td></tr><tr><td>Trypsin</td><td>Merck</td><td>T0303</td></tr><tr><td>Hydrofluoric acid 48%</td><td>Merck</td><td>1.00334.0500</td></tr><tr><td>Mutanolysin</td><td>Merck</td><td>M9901</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452882</td></tr></tbody></table>

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<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Ammonium formate</td><td>Fluka</td><td>17843</td></tr><tr><td>Methanol HPLC LC-MS grade</td><td>VWR Chemicals</td><td>83638</td></tr><tr><td>Qubit™ RNA High Sensitivity</td><td>ThermoScientific</td><td>Q32852</td></tr><tr><td>Pmel</td><td>New England Biolabs</td><td>R0560</td></tr><tr><td>RNAPro solution</td><td>MP Biomedicals</td><td>116055050</td></tr><tr><td>Chloroform:Isoamyl alcohol 24:1</td><td>Merck</td><td>C0549</td></tr><tr><td>Absolute ethanol</td><td>VWR</td><td>20821.296</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Invitrogen</td><td>10977023</td></tr><tr><td>TURBO DNA-free Kit</td><td>Invitrogen</td><td>AM1907</td></tr></tbody></table>

Oligonucleotides

See Table S5

Recombinant DNA

<table><tr><td>pMSR0</td><td>Peltier et al.<sup>35</sup></td><td>N/A</td></tr><tr><td>p226: pMSR0 derivative for CD2963 (ldt<sub>CD1</sub>) deletion</td><td>This study</td><td>N/A</td></tr><tr><td>p227: pMSR0 derivative for CD2713 (ldt<sub>CD2</sub>) deletion</td><td>This study</td><td>N/A</td></tr><tr><td>p261 pMSR0 derivative for CD3007 (ldt<sub>CD3</sub>) deletion</td><td>This study</td><td>N/A</td></tr></table>

Deposited data

<table><tr><td>LC-MS/MS data</td><td>GLYCOPOST repository</td><td>GPST000426</td></tr><tr><td>RNA-seq data</td><td>NCBI SRA</td><td>PRJNA1107170</td></tr><tr><td>Whole genome sequencing data</td><td>NCBI SRA</td><td>PRJNA1107170</td></tr></table>

Software and algorithms

<table><tr><td>Galaxy Europe</td><td>Galaxy<sup>36</sup></td><td>https://usegalaxy.eu/</td></tr><tr><td>Galaxy Pasteur</td><td>Galaxy<sup>36</sup></td><td>https://galaxy.pasteur.fr/</td></tr><tr><td>MicrobeJ v 5.13l</td><td>Rueden et al.,<sup>37</sup> Ducret et al.<sup>38</sup></td><td>https://www.microbej.com/</td></tr><tr><td>CoreLDRAW X8</td><td>CorelDraw</td><td>https://www.coreldraw.com/fr/</td></tr><tr><td>Thermo Xcalibur</td><td>ThermoFisher</td><td>https://www.thermofisher.com/fr/fr/home.html</td></tr><tr><td>Skyline open-source software</td><td>Pinot et al.<sup>39</sup></td><td>https://skyline.ms/project/home/begin.view?/</td></tr></table>

EXPERIMENTAL MODELS AND STUDY PARTICIPANT DETAILS

Bacterial strains and growth conditions

C. difficile strain 630Δ*erm*, a commonly used spontaneous erythromycin-sensitive derivative of the strain 630, and mutant strains Δ*ldt*<sub>CD1</sub>, Δ*ldt*<sub>CD2</sub>, Δ*ldt*<sub>CD3</sub>, Δ*ldt*<sub>CD1</sub>Δ*ldt*<sub>CD2</sub>, Δ*ldt*<sub>CD1</sub>Δ*ldt*<sub>CD3</sub>, Δ*ldt*<sub>CD2</sub>Δ*ldt*<sub>CD3</sub> and Δ*ldt*<sub>CD1</sub>Δ*ldt*<sub>CD2</sub>Δ*ldt*<sub>CD3</sub> (ΔΔΔ*ldt*) were grown in an anaerobic Jacomex workstation with an atmosphere of 5% H₂, 5% CO₂ and 90% N₂. C. difficile strains were cultured in Brain Heart Infusion broth (BHI) or Sporulation Medium for Clostridium difficile (SMC, 9% Bacto peptone, 0.5% proteose peptone, 0.15% Tris base, 0.1% ammonium sulphate).

Plasmids were maintained in E. coli strain NEB10β and transformed using standard procedures. E. coli HB101 carrying the plasmid RP4 was used for plasmid conjugation with C. difficile strains.⁴⁰ The E. coli strains were cultured in Luria Bertani broth (LB) supplemented with chloramphenicol at 15 µg/mL or 100 µg/mL ampicillin when required, and were grown aerobically at 37°C.

The growth was followed by optical density reading at 600 nm. Growth curves were performed in 96-well plates in BHI medium in the absence or the presence of antibiotics (1/2 MIC), with a starting culture at a normalized OD<sub>600</sub> ≈ 0.05, under anaerobic conditions at 37°C, in Stratus Plate Reader (Cerillo). The OD<sub>600</sub> value for each well was recorded every 30 min.

METHOD DETAILS

Construction of the C. difficile *ldt* mutants

All oligonucleotides used in this study are listed in Table S5. The C. difficile *ldt* deletion mutants were created using a toxin-mediated allele exchange method.³⁵ Briefly, ~800 bp homology arms flanking the region to be deleted were amplified by PCR from C. difficile 630Δ*erm*. Purified PCR products were cloned into the Pmel site of the pseudo-suicide allele-coupled exchange (ACE) vector pMSR0
using NEBuilder HiFi DNA Assembly (New England Biolabs). The resulting plasmids, listed in the key resources table, were transformed into *E. coli* strain NEB10β (New England Biolabs) and all inserts were verified by sequencing. Plasmids were then transformed into *E. coli* HB101(RP4) and transferred by conjugation into the appropriate *C. difficile* strains. Transconjugants were selected on BHI supplemented with *Clostridioides difficile* Selective Supplement (CDSS; Oxoid), and 7.5 µg/mL thiamphenicol. Allelic exchange was performed as described.³⁵ All strains were confirmed by locus amplification. The triple mutant was further confirmed by whole-genome sequencing.

## Whole genome sequencing

*C. difficile* strains were grown in BHI for 24h. Cells were harvested and genomic DNA was isolated using NucleoSpin Microbial DNA Mini kit for DNA (Macherey-Nagel). Bacterial genomes were sequenced at Plasmidsaurus (https://www.plasmidsaurus.com/) using the long-read sequencing technology from Oxford Nanopore Technologies (ONT). Using Galaxy Europe, quality controls were performed with FastQC and nanoplot and adapter trimming was performed with Porechop. The resulting reads were mapped to the reference genome using Map with minimap2. Small indels (<50 bp) and SNPs were identified using Clair3, bcftools norm and SnpSift Filter. Large indels were identified using CuteSV. Raw sequence files were deposited to the NCBI Sequence Read Archive (SRA) BioProject PRJNA1107170.

## Fluorescence microscopy

The sample preparation for fluorescence microscopy was carried out under anaerobic conditions. *C. difficile* strains were cultured in BHI. When required, 1 mL culture was incubated with HADA at a final concentration of 10 µM (λem ≈ 450 nm, Tocris Bioscience)¹⁹ for 10 min and washed 3X with anaerobic PBS. Cells were spotted on GeneFrame slides with 1.5% agarose patches. Microscopy was carried out with spinning disk confocal microscopy, using an Inverted Eclipse Ti-E (Nikon) equipped with CSU-X1-A1, Nipkow Spinning Disk confocal system (Yokogawa) and ORCA-Flash4.0 LT CMOS camera (Hamamatsu). Data and statistical analysis were done with MicrobeJ version 5.13l plugin for ImageJ.³⁷,³⁸ Recognition of cells was limited to 1 µm² minimum and 1 - 16 µm length. Cells with defective detection were excluded from analysis. Fluorescent intensity profiles of contour and medial were used for analysis of 2 independent biological replicates. Representative pictures were prepared for publication in CorelDRAW X8 (Corel).

## Spore purification

Overnight cultures of *C. difficile* ΔΔΔ/dt and parental strains grown in BHI were spread out on twelve SMC agar plates for sporulation.⁴¹ After 7 days of anaerobic incubation at 37°C, cells and spores were harvested in 2 ml of ice-cold sterile water. Crude suspensions were washed 10 times with ice-cold sterile water and spores were purified with a HistoDenz gradient 20-50%.

## Purification and structural analysis of PG

Vegetative PG was extracted from *C. difficile* strains as previously described with some modifications.¹⁴ *C. difficile* cultures were grown to OD₆₀₀ ≈ 1.0 at 37°C in BHI with the addition of cefoxitin (64 µg/mL), ceftriaxone (32 µg/mL), meropenem (1 µg/mL) or imipenem (2 µg/mL), when required. Cells were harvested by centrifugation at 5000g for 10 min and processed in a FastPrep apparatus (MP Bioscience) for 30 s at 4 m/s to break the cells. For spore PG extraction, purified spores were additionally incubated twice in decoating buffer (50 mM Tris-HCl pH8.0, 8M Urea, 1% sodium dodecyl sulfate (SDS), 50 mM DTT) for 1h at 37°C. Samples were harvested by centrifugation at 17000g for 10 min.

Both cell and spore pellets were resuspended in cold H₂O, boiled for 10 min, cooled again, and centrifuged at 17000g for 10 min. The cells were then boiled in 10% SDS, followed by boiling in 4% SDS and washed 10 times with H₂O. The insoluble material was then treated with pronase for 90 min at 60°C, followed by incubation with DNase, RNase, Lipase and trypsin for 20h at 37°C to purify the cell wall. Finally, the samples were incubated in 48% hydrofluoric acid at 4°C for 16 h to remove wall polysaccharides. Purified PG was digested with mutanolysin (Sigma), and the soluble muropeptides were reduced with sodium borohydride. Muropeptides were analyzed by LC-MS/MS with an UHPLC instrument (Vanquish Flex, Thermo Scientific) connected to a Q-Exactive Focus mass spectrometer (Thermo Fisher Scientific) fitted with an H-ESI electrospray source (facilities located at ChemSyBio, Micalis, INRAE, Jouyen-Josas). They were separated by reverse phase chromatography with a ZORBAX Eclipse Plus C18 RRHD column (100 by 2.1mm; particle size, and 1.8µm; Agilent Technologies) at 50°C using 10 mM ammonium formate buffer (pH 4.6) and a 20 min linear gradient from 0 to 20% methanol at a flow rate of 0.3 ml/min. Mass analysis was performed in positive mode with an acquisition range of 380 - 1400 m/z at resolution 17,500. The Q-Exactive mass spectrometer was operated with capillary voltage at 3.5 kV and a capillary temperature set at 320°C. MS2 was performed in an acquisition range of 160-1600 m/z with an AGC target at 2.10⁵ with HCD collision anode at energy 25. Data were acquired with the Qual Browser suite (Thermo Xcalibur).

Muropeptides were identified from their m/z values and MS/MS spectra when required. Their relative amount was determined using Skyline open-source software according to the Small Molecule Quantification workflow.³⁹ A transition list was created manually containing all the muropeptides identified by their retention times and including the different adducts and charge states detected by MS. Extraction ion chromatograms (EICs) and peak integration were realized after importing raw data from Xcalibur files. The relative amount (%) of each muropeptide was calculated as the ratio between the sum of EIC areas assigned to the muropeptide and the sum of EIC areas assigned to all identified muropeptides.
The cross-linking index (CI) was calculated according to Glauner as follows: $CI = (1/2 Σdimers + 2/3 Σtrimers)/Σall muropeptides.$^{42}$
The relative amount of muropeptides with a certain peptide side chain (X= Di, Tri, Tetra, Penta) with a free terminal carboxyl group (acceptor chain in multimers) was calculated according to Glauner as follows: percentage (X) = $(Σmonomers(X) + 1/2Σdimers + 1/3Σtrimers)/Σall muropeptides.$^{42}

## Minimum inhibitory concentration (MIC) determination

For the determination of the MIC, strains were grown until OD$_{600}$ of 1.0 and inoculated to a starting optical density 0.05 in 200 µL BHI medium supplemented with the required antibiotic range (amoxicillin, oxacillin, cefoxitine, ceftriaxone, imipenem or meropenem) on 96-well plates. Cultures were incubated at 37°C for 24h. MICs for each strain were determined as the lowest concentration without visible growth, at least in three independent experiments.

## RNA sequencing

For total RNA extraction, *C. difficile* strains were grown in 20 mL BHI medium with 32 µg/mL ceftriaxone, until an optical density of 1.0. Total RNA was isolated as previously described.⁴³ Quantification of the RNA samples was performed using the Qubit™ RNA High Sensitivity (ThermoScientific). Samples were sent for RNA sequencing by Novogene Prokaryotic RNA Sequencing services (Novogene). Analysis of the raw FastQ files was performed with Galaxy Pasteur.³⁶ Quality control of the raw data was assessed using FastQC and raw data were then subjected to trimming and filtering with AlienTrimmer.⁴⁴ Sequencing reads were aligned to *C. difficile* strain 630 genome (NC_009089.1) with the Bowtie2 software using default parameters.⁴⁵ SARTools DESeq2 was used to perform normalization and differential analysis using values of the 630Δerm wild-type strain as a reference for reporting the expression data of the ΔΔΔ/dt strain. Genes were considered differentially expressed if they had a ≥ two-fold increase or decrease in expression and an adjusted *P* value (*q* value) ≤ 0.05. Assignment of the functional orthologues (K number) and KEGG pathway was performed automatically using BlastKOALA,⁴⁶ and was then manually edited. RNA-seq raw sequence reads were deposited to the NCBI Sequence Read Archive (SRA) BioProject PRJNA1107170.

## QUANTIFICATION AND STATISTICAL ANALYSES

Statistical analyses were performed using GraphPad Prism 10. Data are presented as the mean values ± SEM of at least three independent experiments with the following exceptions: in the *Figures 2B–2D*, graphs represent mean ± SEM of two independent experiments for the mutant strains $Δdt_{Cd1}$, $Δdt_{Cd2}$, $Δdt_{Cd3}$, $Δdt_{Cd1}Δdt_{Cd2}$, $Δdt_{Cd1}Δdt_{Cd3}$, $Δdt_{Cd2}Δdt_{Cd3}$, and in the *Figures S2C–S2F*, two independent experiments were analysed for *C. difficile* 630Δerm and ΔΔΔ/dt. All the statistical details of experiments, including the number of independent replicates *n*, what *n* represents and the statistical tests used, can be found in the figure legends. Statistical analyses were performed using one-way ANOVA or two-way ANOVA, both followed by a Dunnett's multiple comparisons test. For all figures, *P* values are represented as follows: *P* < 0.05, **P* < 0.01, ***P* < 0.001, ****P* < 0.0001.