Jeanne Malet-Villemagne

Yucheng Liang

Laurent Evanno

Sandrine Denis- Quanquin

Jean-Emmanuel Hugonnet

Michel Arthur

Claire Janoir

Thomas Candela
email: thomas.candela@universite-paris-saclay.fr

Polysaccharide II surface anchoring, the Achilles' heel of Clostridioides difficile

Cell wall glycopolymers (CWPGs) in Gram-positive bacteria have been reported to be involved in several bacterial processes. These polymers, pillars for proteins and S-layer, are essential for the bacterial surface set-up, could be essential for growth, and, in pathogens, participate most often in virulence. CWGPs are covalently anchored to the peptidoglycan by Lcp proteins that belong to the LytR-CpsA-PSr family. This anchoring, Cell wall glycopolymers (CWGPs) in gram-positive bacteria have been reported to be involved in several bacterial processes. The CWGPs anchoring to the peptidoglycan is important for growth and virulence. We set up the first conditional-lethal mutant method in C. difficile to study LcpA and LcpB involved in the anchoring of the CWPGs to the peptidoglycan. This study offers new tools to reveal the role of essential genes in C.

difficile. LcpA and LcpB activity was shown to be essential, suggesting that they are potential new targets to combat C. difficile infection. In this study, we also showed that there is a competition between the polysaccharide II synthesis pathway and the peptidoglycan synthesis that probably exists in other Gram-positive bacteria. A better understanding of these mechanisms allows us to define the Lcp proteins as a therapeutic target for a potential design of a novel antibiotic against pathogenic Grampositive bacteria.

important for growth, was reported as essential for some bacteria such as Bacillus subtilis, but the reason why CWGPs anchoring is essential remained unknown. We studied LcpA and LcpB of Clostridioides difficile (C. difficile) and showed that they have a redundant activity. To delete both lcp genes, we set up the first conditional-lethal mutant method in C. difficile and showed that polysaccharide II (PSII) anchoring at the bacterial surface is essential for C. difficile survival. In the conditional-lethal mutant, C. difficile morphology was impaired, suggesting that peptidoglycan synthesis was affected.

Because Lcp proteins are transferring CWPGs from the C55-undecaprenyl phosphate, also needed in the peptidoglycan synthesis process, we assumed that there was a competition between the PSII and the peptidoglycan synthesis pathways. We confirmed that UDP-MurNAc-pentapeptide precursor was accumulated, showing that peptidoglycan synthesis was blocked. Our results provided an explanation for the essentiality of PSII anchoring in C. difficile and suggest that the essentiality of the anchoring of CWPGs in other bacteria can also be explained by the blocking of peptidoglycan synthesis. To conclude, our results suggest that Lcps are potential new targets to combat C. difficile infection.

Importance Introduction

In recent years, there has been an increasing interest in cell wall glycopolymers (CWGPs) in Gram-positive bacteria for their role in bacterial physiology and pathogenicity. These polymers represent up to 50% of the dry weight of the cell wall [START_REF] Neuhaus | A continuum of anionic charge: structures and functions of D-alanylteichoic acids in gram-positive bacteria[END_REF].

CWGPs are covalently linked to peptidoglycan (PG). They support surface proteins (like Cwp in Clostridioides difficile or SLH in Bacillus subtilis) that are non-covalently anchored at the very end of CWGPs [START_REF] Mesnage | Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation[END_REF][START_REF] Willing | Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII[END_REF]. Most of them are essential and involved in cell division and cell shape maintenance (for review (4)). In pathogens, they are essential for virulence [START_REF] Weidenmaier | Lack of wall teichoic acids in Staphylococcus aureus leads to reduced interactions with endothelial cells and to attenuated virulence in a rabbit model of endocarditis[END_REF][START_REF] Weidenmaier | DltABCD-and MprF-mediated cell envelope modifications of Staphylococcus aureus confer resistance to platelet microbicidal proteins and contribute to virulence in a rabbit endocarditis model[END_REF][START_REF] Collins | Staphylococcus aureus strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice[END_REF][START_REF] Fisher | The dltABCD operon of Bacillus anthracis sterne is required for virulence and resistance to peptide, enzymatic, and cellular mediators of innate immunity[END_REF][START_REF] Spears | Listeria monocytogenes wall teichoic acid decoration in virulence and cell-to-cell spread[END_REF][START_REF] Meireles | Listeria monocytogenes Wall Teichoic Acid Glycosylation Promotes Surface Anchoring of Virulence Factors, Resistance to Antimicrobial Peptides, and Decreased Susceptibility to Antibiotics[END_REF][START_REF] Kharat | Different pathways of choline metabolism in two choline-independent strains of Streptococcus pneumoniae and their impact on virulence[END_REF]. They are therefore thought to be targets for the conception of new antimicrobial molecules [START_REF] Lee | TarO-specific inhibitors of wall teichoic acid biosynthesis restore β-lactam efficacy against methicillin-resistant staphylococci[END_REF][START_REF] Sewell | Taking aim at wall teichoic acid synthesis: new biology and new leads for antibiotics. 1[END_REF].

CWGPs anchoring is of major importance for the physiology of Gram-positive bacteria. This process was shown to be done by proteins belonging to the LytR-CpsA-PSr (LCP) family. Members of this protein family are widespread in the bacterial world as they were identified in eight different bacterial phyla [START_REF] Hübscher | Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family[END_REF]. The Lcp enzymes transfer CWGPs from a lipid carrier, the C 55 -undecaprenyl phosphate (C 55 P) to the nascent or mature PG [START_REF] Atilano | Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus[END_REF][START_REF] Schlag | Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl[END_REF][START_REF] Bhavsar | The TagB protein in Bacillus subtilis 168 is an intracellular peripheral membrane protein that can incorporate glycerol phosphate onto a membrane-bound acceptor in vitro[END_REF][START_REF] Andre | Fluorescence and atomic force microscopy imaging of wall teichoic acids in Lactobacillus plantarum[END_REF][START_REF] Rausch | Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus[END_REF]. Lcp proteins are therefore key players in bacterial surface assembly. In Gram-positive bacteria, several copies of lcp genes are often observed but the gene products can have distinct catalytic activities. For example, in B. subtilis, three Lcp proteins have been identified and one of them (TagU) has a stronger activity than the two others [START_REF] Gale | B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro[END_REF]. In M. tuberculosis, the only Lcp is essential [START_REF] Malm | In vivo virulence of Mycobacterium tuberculosis depends on a single homologue of the LytR-CpsA-Psr proteins[END_REF]. Finally, in Staphylococcus aureus (S. aureus), LcpA is the main transferase of teichoic acids whereas LcpC is dedicated to the capsular polysaccharides transfer [START_REF] Rausch | Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus[END_REF]. The Lcp proteins are at least partially redundant in B. subtilis [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF], S. aureus [START_REF] Chan | Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium[END_REF][START_REF] Chan | The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes[END_REF], and Streptococcus pneumoniae [START_REF] Yother | Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation[END_REF][START_REF] Eberhardt | Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae[END_REF]. Besides, the Lcp proteins are good targets for specific inhibitors development because their soluble catalytic domain is extracellular (accessible to immune system cells) and they have no homologs in mammals [START_REF] Hübscher | Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family[END_REF]. In C. difficile, Lcps were studied individually [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF], but their redundancy and essentiality for survival have never been assessed experimentally.

Clostridioides difficile (previously known as Clostridium difficile) is a Grampositive, motile, strictly anaerobic, and spore-forming enteric pathogen. C. difficile infections (CDIs) are a primary cause of nosocomial diarrhea and antibiotic-associated colitis [START_REF] Bartlett | Clinical and laboratory observations in Clostridium difficile colitis[END_REF]. Besides, the incidence and the number of severe clinical forms have been increasing in recent years, due to emerging hypervirulent and antibiotic-resistant strains (including C. difficile ribotype 027) [START_REF] Zilberberg | Increase in adult Clostridium difficile-related hospitalizations and case-fatality rate, United States, 2000-2005[END_REF]. Consequently, C. difficile is considered an urgent threat to public health by the Centers for Disease Control and prevention [START_REF]Antibiotic resistance threats in the United States, 2019[END_REF].

Moreover, multiple resistance mechanisms to currently used antibiotics, often mediated by plasmid acquisition [START_REF] Pu | Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile[END_REF][START_REF] Boekhoud | Plasmid-mediated metronidazole resistance in Clostridioides difficile[END_REF], are observed and cause concerns about future treatment options for CDI management. Therefore, developing new strategies and discovering new bacterial targets is necessary.

In C. difficile, three CWPGs have been identified: two teichoic acids (TA) anchored in the peptidoglycan (polysaccharides I and II, [START_REF] Ganeshapillai | Clostridium difficile cellsurface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units[END_REF]) and one lipoteichoic acid (LTA) anchored directly in the membrane [START_REF] Reid | Structural characterization of surface glycans from Clostridium difficile[END_REF]. Only the polysaccharide II (PSII) and the LTA are conserved among C. difficile strains. All the biosynthesis genes of these glycopolymers are encoded in a single locus of the chromosome. All the genes predicted to encode enzymes involved in PSII synthesis are reported to be essential for bacterial survival [START_REF] Willing | Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII[END_REF][START_REF] Dembek | High-throughput analysis of gene essentiality and sporulation in Clostridium difficile[END_REF], but surprisingly not lcpA and lcpB genes encoding PSII anchoring proteins [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF]. Note that Cwp proteins, including the S-layer protein SlpA, are noncovalently linked to the PSII [START_REF] Willing | Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII[END_REF].

In this study, we focused on PSII. Its biosynthesis is predicted to be initiated by the transferase CD2783, transferring the first sugar unit (UDP-Glucose) on the C 55 P lipid carrier. Then, several cytoplasmic glycosyltransferases catalyze the transfer of sugar units on the chain. When finished, the chain is flipped outside the cell by the flippase MviN [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF] and transferred to the pre-existing chains to form a C 55 P-polymerized PSII molecule. PSII is then transferred from the lipid carrier to the PG by LcpA and LcpB [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF].

In this work, we studied the two Lcps of C. difficile to evaluate the importance of a correct polysaccharide anchoring in the physiology of the bacterium. We wanted to determine if the lcp genes were essential for survival.

Results

Construction of lcp single mutants

To facilitate the screen of the allelic exchange technique in C. difficile [START_REF] Peltier | Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile[END_REF], we decided to replace the open reading frame of lcpA and lcpB with the open reading frame of the catP gene expressing the thiamphenicol resistance. To that aim, we constructed the pJV10 vector that harbors an ermB gene (Figure S1) conferring erythromycin resistance.

In the genome of the 630 strain, two copies of the ermB gene are found. In contrast, the 630Δerm harbors only one ermB gene [START_REF] Hussain | Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn916DeltaE enters the genome of this strain at multiple sites[END_REF], which is usually not expressed. However, we were unable to conjugate the pJV10 plasmid in the 630Δerm that became, in these conditions, resistant to erythromycin, probably because the remaining ermB gene was sufficiently expressed during the conjugation process. Therefore, we chose to construct a "true" 630Δerm by deleting both ermB genes directly from the clinical 630 strain and obtained the JMV1 strain.

We first deleted lcpA and lcpB separately in the JMV1 strain using the allelic exchange method [START_REF] Peltier | Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile[END_REF]. Thanks to the presence of the catP gene, after the selection of the first crossing over [START_REF] Peltier | Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile[END_REF], a simple restreak on a petri dish in the presence of thiamphenicol allows the identification of potential mutant clones. The JMV3 (∆lcpA) strain was easily obtained (21 mutant clones were thiamphenicol resistant out of 25) whereas the JMV4 (∆lcpB) strain was quite hard to get (3 mutant clones were thiamphenicol resistant out of 187), suggesting that lcpB plays an important role for C. difficile growth.

lcpA and lcpB are redundant

We first confirmed that the lcpB mutant (JMV4) showed a growth defect (Figure S2). Then, we observed the morphology of the mutant cells in classical optic microscopy (Figure 1A) and measured the cell length and width (Figure 1C and 1D), allowing us to determine a percentage of "normal morphology" (Figure 1B). Contrary to the ∆lcpA mutant bacteria (JMV3) whose morphology is normal, almost 35% of the ∆lcpB (JMV4) cells were curved or inflated (Figures 1 andS3). Cells were also significantly longer and thicker than the JMV1 cells (Figures 1C and1D). These observed morphological and growth defects in the absence of lcpB confirmed the previous results obtained by Vedantam et al. [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF] and suggest that LcpB plays a major role in anchoring the PSII to the peptidoglycan. Besides, we observed that bacterial morphology and growth were restored in the strains JMV4 + pMEZ12 (complementation plasmid bearing lcpA, named thereafter plcpA for simplification) and JMV4 + pJV21 (complementation plasmid bearing lcpB, named thereafter plcpB for simplification), as shown in Figures 1A andS2. Indeed, the abnormal cell ratio was reduced to 4% and 6% when complementation with lcpA or lcpB was introduced (Figure 1B). These results suggest that LcpA can compensate for the absence of LcpB in anchoring the PSII to the peptidoglycan and that both Lcp proteins have redundant functions in C. difficile.

Considering the absence of phenotype in the JMV3 strain, we wondered if lcpA was expressed and we assessed the expression of lcpA and lcpB by a measurement of the promoter activity (by beta-glucuronidase assay). As shown in Figures 2A and2B, lcpA and lcpB had constitutive expression, respectively with a mean of 230 and 35 Miller units, but lcpA is transcribed at a higher level than lcpB. This result suggested, that even if transcribed at a low level, lcpB is important for cell growth and morphology.

The ∆lcpA (JMV3) and ∆lcpB (JMV4) mutant strains present a normal surface protein profile but an altered PSII layer Because Cwp proteins are non-covalently anchored to the PSII, we analyzed the S-layer content (Figure S4). We found no differences between the CWP proteins of the parental strain and the single mutants. To assess the presence of PSII at the bacterial surface, we purified the PSII, checked that it was non-contaminated with LTA by NMR (Figure S5), and coupled it with BSA. After immunization, we obtained specific antibodies able to recognize the PSII (Figure S6). Using a super-resolution microscope, the JMV1 parental strain showed a homogenous and continuous layer of PSII along the bacterium. In contrast, both JMV3 and JMV4 mutant strains showed an altered PSII layer (Figure 3). The JMV3 cells presented a holed layer of PSII. The JMV4 mutant strain presented a smooth PSII layer. The alteration of the PSII deposition at the surface was present in both mutants but the PSII layer was differently altered in the JMV3 and JMV4 strains, suggesting that even though redundant in activity, LcpA and LcpB have slightly different roles in PSII anchoring at the bacterial surface.

PSII anchoring is essential for C. difficile survival

Once the single mutants were obtained and their phenotypes confirmed, we tried to get a double mutant strain to assess the essentiality of the PSII anchoring for C. difficile survival. The first strategy was to use our improved allelic exchange method using the pJV13 plasmid and the C. difficile JMV1 strain. Despite the facilitated screening of mutants, we failed to isolate a double lcp mutant over the 450 clones tested. This result suggested that deleting both lcpA and lcpB genes was not possible, maybe because ofthe essentiality of bothLcpA and LcpB.

To assess the essentiality of lcp genes in C. difficile, we elaborated a new strategy based on the construction of a conditional-lethal mutant (Figure S7). The first step was to insert an extra copy of lcpB under the control of a P tet promotor in the ermB locus of the 630, to mimic the JMV1 strain by removing both ermB genes, giving rise to the JMV2 strain. The second step was to perform the deletion of both lcpA and lcpB in the JMV2 strain using pJV13 plasmid in the presence of 100 ng.mL -1 of anhydrotetracycline (ATc).

We obtained the conditional-lethal mutant strain JMV6 that was not able to grow without induction of the additional copy of lcpB (Figures 4A andS8). To confirm this phenotype, conditional-lethal mutant strain (JMV6) was grown in the presence of 10 or 50 ng.mL -1 of ATc and then plated on a Petri dish with no or up to 250 ng.mL -1 (Figure 4A). No growth was observed on plates at 10 ng.mL -1 ATc or less, showing that the presence of at least one lcp is essential for C. difficile growth. In liquid culture, conditional-lethal mutant strain (JMV6) in the presence of 10 ng.mL -1 had an impaired growth that was restored by adding 50 ng.mL -1 of ATc (Figure 4B). Without ATc, growth was restored when lcpA was present (on plcpA plasmid) confirming the redundancy of Lcp activity. We also assessed the morphology using microscopy and confirmed that the conditional-lethal mutant strain (JMV6) grown with 10 ng.mL -1 of ATc had a marked phenotype with ellipsoid cells shorter and thicker than the JMV1 cells (Figure 5). In the presence of 50 ng.mL -1 of ATc, some bacilli were curved and long but the rod shape was restored with comparable cell width and increased cell length compared to JMV1 cells (Figure 5). Finally, the addition of plcpA or plcpB fully restored the bacterial shape, similarly to the controls (JMV1 and JMV2 strains). Our results showed that the absence of lcpA and lcpB is lethal for C. difficile and suggested that PSII anchoring is essential for C. difficile growth. In addition, this result suggested that only lcpA and lcpB are involved in PSII anchoring.

PSII remains at the bacterial surface in the JMV6 strain

To analyze the localization of PSII at the bacterial surface when its anchoring is impaired due to the limitation of LcpA and LcpB, the conditional-lethal mutant strain (JMV6) was cultured with 10 ng.mL -1 or 50 ng.mL -1 of ATc (Figure 6). We used the JMV2 strain as a control, which has a second copy of lcpB (P tet -lcpB copy at the ermB locus). This strain has a similar phenotype to the JMV1 strain confirming that overexpression of lcpB, due to the induction of the second copy, does not affect PSII anchoring and bacterial morphology. In the conditional-lethal mutant strain (JMV6), we confirmed that a low induction of lcpB (10 ng.mL -1 of ATc) leads to ellipsoid cells. The rod shape was restored in the presence of 50 ng.mL -1 of ATc with or without lcpA. Moreover, PSII was still localized at the bacterial surface of the JMV6 strain in the presence of 10 ng.mL -1 of ATc (Figure 6, JMV6 (ATc 10)). This result was surprising because, according to the previous study of Chu et al. [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF], PSII was expected to be found in the supernatant fraction. Our results suggested that the PSII, after its synthesis was still anchored to its lipid carrier at the plasmic membrane, in accordance with the models [START_REF] Gale | B. subtilis LytR-CpsA-Psr Enzymes Transfer Wall Teichoic Acids from Authentic Lipid-Linked Substrates to Mature Peptidoglycan In Vitro[END_REF][START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF].

Part of the surface PSII and Cwp proteins is released in the JMV6 strain

To assess the impact of a defect of PSII anchoring to PG, we analyzed the presence of PSII at the bacterial surface and in the supernatant by dot blot analysis (Figure 7). In the JMV1 parental strain and the JMV2 control strain (P tet -lcpB), the PSII was found in the bacterial fraction (pellet), suggesting that it was only associated with the bacterial surface. The same result was observed for the single lcp mutant strains JMV3 and JMV4. Conversely, in the conditional-lethal lcp mutant (JMV6) with 10 ng.mL -1 of ATc, PSII was found at the bacterial surface and released in the culture supernatant. This release of PSII is decreased in the presence of 50 ng.mL -1 of ATc. The phenotype is completely restored in the conditional-lethal mutant strain (JMV6) in the presence of lcpA and 50 ng.mL -1 of ATc.

Because the PSII was released in the supernatant, we assessed whether the Cwp proteins, which are non-covalently linked to the PSII, were also found in the supernatant.

We showed that the Cwp amount was decreased at the bacterial surface of the conditional-lethal mutant strain (JMV6) in the presence of 10 ng.mL -1 ATc induction, in comparison with the JMV1, JMV2, and JMV6+lcpA strains (Figure 8A). Cwp proteins of the conditional-lethal mutant strain (JMV6) strain in the presence of 10 ng.mL -1 were found in the supernatant. In comparison, in the presence of 50 ng.mL -1 , Cwp proteins from the JMV6 strain were more abundant at the bacterial surface. To further characterize which proteins were concerned, we performed a western blot analysis.

These analyses allowed us to identify two proteins of the Cwp family, Cwp66 and SlpA, in the supernatant of the conditional-lethal mutant strain (JMV6) strain (Figures 8D and8F). Accordingly, Cwp66 was absent from the surface protein extracts (Figure 8C) and SlpA was found in a lower quantity than in other strains (Figure 8E). It is to note that SlpA precursor (uncleaved) was found in the conditional-lethal mutant strain (JMV6), suggesting a maturation defect. We analyzed the autolysis profile of all strains to investigate why the PSII and the Cwp proteins were found in the supernatant (Figure 9). We found that the single lcp mutants JMV3 and JMV4 autolyzed more rapidly than the parental strain (Figure 9A) and that this phenotype was absent when they were complemented with either plcpA or plcpB. The conditional-lethal mutant JMV6 autolyzed also more rapidly than the JMV1 strain (Figure 9B). Again, the impaired phenotype was fully restored in the presence of 50 ng.mL-1 of ATc and lcpA. These results suggested that the conditional-lethal mutant strain (JMV6) is lysing more rapidly than the parental strain, explaining the partial release of the PSII into the culture supernatant.

Cytoplasmic PG precursors accumulate in response to impaired PSII anchoring to PG Because the PSII is attached to the C 55 P carrier during its biosynthesis and until an Lcp protein anchors it to the peptidoglycan, we hypothesized that the PSII transfer impairment from the C 55 P carrier to the peptidoglycan may limit the availability of this lipid carrier for peptidoglycan synthesis. The extraction of cytoplasmic peptidoglycan precursors was performed for JMV1, conditional-lethal mutant strain JMV6 (10 ng.mL -1 ATc), and conditional-lethal mutant strain JMV6 + plcpA (50 ng.mL -1 ATc) (Figure 10). In the JMV1 strain, only peak 1 was found (Figure 10A). In the two other tested strains (Figures 10B and10C), peak 1 and peak 2 were found. Mass spectrometry analyses (Figure 10D) indicated that the precursor in peak 1 was UDP-MurNAc-pentapeptide.

Analysis of the precursor in peak 2 by tandem mass spectrometry indicated that it differed from UDP-MurNAc-pentapeptide by the amidation of the side-chain carboxyl of the diaminopimelyl (DAP) residue located at the 3 rd position of the pentapeptide stem. This amidation, attributed to AsnB, was only reported once, when C. difficile was grown in the presence of vancomycin at a sublethal concentration [START_REF] Ammam | AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin[END_REF]. A third peak (Figure 10B andC) was not identified. UDP-MurNAc-pentapeptide was 21-fold more abundant in JMV6 grown in the presence of 10 ng.mL -1 of ATc than in the parental JMV1 strain.

The accumulation of UDP-MurNAc-pentapeptide was less abundant (6 fold instead of 21 fold) in the JMV6 + plcpA strain, in the presence of 50 ng.mL -1 of ATc. These results establish that impaired PSII anchoring to peptidoglycan results in the accumulation of the UDP-MurNAc-pentapeptide peptidoglycan precursor. This accumulation is likely to result from a limited availability of the C 55 P lipid carrier for peptidoglycan synthesis due to its sequestration in lipid-linked PSII precursors.

Discussion

In this study, we characterized LcpA and LcpB as responsible for PSII anchoring to C. difficile PG. In addition, we showed that the activity of these proteins is essential for the viability of C. difficile probably because of an interference with the PG synthesis.

In well-studied Gram-positive models like B. subtilis, S. pneumoniae, and S. aureus, lcp genes are found in multiple copies in the genome and are at least partially redundant [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF][START_REF] Chan | Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium[END_REF][START_REF] Chan | The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the LytR-CpsA-Psr (LCP) family of enzymes[END_REF][START_REF] Yother | Capsules of Streptococcus pneumoniae and other bacteria: paradigms for polysaccharide biosynthesis and regulation[END_REF][START_REF] Eberhardt | Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae[END_REF]. Our study confirmed that growth of a lcpB mutant strain is associated with morphological defects, contrary to a lcpA mutant strain [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF]. lcpB appears then to be more important than lcpA, yet lcpB is expressed at a lower level than lcpA. The morphological and growth defects of the lcpB mutant were restored by overexpression of lcpA. The overexpression may localize LcpA differently than in the parental strain, allowing the complementation by compensating the absence of LcpB at the bacterial surface and suggests that LcpA and LcpB have partially redundant functions. Similarly, in other bacteria, yet redundant in activity, one Lcp has a predominant role, and its absence impacts bacterial physiology more than the others [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF][START_REF] Zilla | LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly[END_REF][START_REF] Schaefer | In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins[END_REF].. Our immunofluorescence study (Figure 3) showed that the PSII layer is altered in both single mutants but differently, suggesting that these distinct phenotypes can be due to a different localization of the two Lcps at the surface. It is to note that LcpB is predicted to have a transmembrane domain and LcpA only has a signal peptide domain (https://www.ebi.ac.uk/interpro/), suggesting that LcpB is localized at the membrane and LcpA is secreted. Since the PSII is linked to the C 55 P at the membrane, a membranous Lcp (LcpB in C. difficile) may be more efficient in transferring it from the C 55 P to PG. In contrast, LcpA should be less efficient because of its lack of an Nterminal transmembrane domain which is untypical among Lcp proteins, since they usually have at least a transmembrane domain [START_REF] Hübscher | Phylogenetic distribution and membrane topology of the LytR-CpsA-Psr protein family[END_REF].

Lcp proteins are phosphotransferases according to Kawai et al. [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF] or peptidoglycan-glycopolymer ligase according to Schaefer et al. [START_REF] Schaefer | In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins[END_REF]. However, in lcp mutants of S. aureus and B. subtilis, CWGPs were found to be released [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF][START_REF] Chan | Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium[END_REF][START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF].

There is a discrepancy between these data and the theoretical CWGP synthesis and transfer of the CWGPs from the C 55 P to the PG. This was explained in S. aureus by the activity of CapA1 that catalyzes the cleavage of the pyrophosphate linkage between the CWGP and the C 55 P, releasing the CWGP into the supernatant in the absence of Lcp proteins. In contrast, in S. pneumoniae [START_REF] Eberhardt | Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae[END_REF] and our study, we reported that the CWGPs were found both in the supernatant and at the bacterial surface. In our work, it is difficult to know whether this PSII localization is due to the presence of a low level of LcpB (JMV6 in the presence of 10 ng.mL -1 of ATc) or if PSII is still anchored to C 55 P carrier at the surface. In C. difficile, one gene encodes a putative protein similar to that of CapA1 from S. aureus (CD630_11190, 19% of identity and 45% of similarity) and none was found in the S. pneumoniae R6 genome. The CD630-11190 putative lipoprotein may have another function than CapA1, but we can't exclude that the observed release of PSII into the supernatant may be due to this protein, together with the observed bacterial lysis (Figure 9). Additionally, we showed that PSII release in the conditional-lethal strain was associated with the release of the Cwp66 and SlpA surface proteins in the supernatant. Indeed, we were able to detect SlpA at the bacterial surface which is the most abundant surface protein in C. difficile, but not Cwp66, suggesting that most of the Cwp proteins are not localized at the bacterial surface anymore. In parallel, we observed that SlpA was only partially matured in the JMV6 strain, suggesting that Cwp84 was not efficient in its cleavage. This defect in SlpA cleavage may be due to the Cwp84 localization that was suggested to be first active when positioned at the surface, then released after an auto maturation, and finally fully active and reassociated to the bacterial surface [START_REF] Chapetónmontes | Localization of the Clostridium difficile cysteine protease Cwp84 and insights into its maturation process[END_REF]. This last step may be missing due to a probable association with the released PSII instead of the bacterial surface, explaining the partial defect in SlpA cleavage.

Blocking indirectly the recycling of C 55 P, for example with cell wall synthesis inhibitors (such as bacitracin and vancomycin), leads to an accumulation of UDP-MurNAc-pentapeptide in the cytoplasm and then bacterial death [START_REF] Hashizume | Tripropeptin C Blocks the Lipid Cycle of Cell Wall Biosynthesis by Complex Formation with Undecaprenyl Pyrophosphate[END_REF]. Because the PSII is predicted to be anchored on the C 55 P lipid carrier during its biosynthesis [START_REF] Willing | Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII[END_REF][START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF] and until it is transferred by Lcp proteins to the PG [START_REF] Rausch | Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus[END_REF], we hypothesized that an impairment in PSII anchoring could lead to a blocking of peptidoglycan biosynthesis through a competition between the C 55 P linked PSII and the synthesis of lipid II that requires free C 55 P. Our results suggest that the sequestration of C 55 P-linked PSII blocks the transfer of the UDP-MurNAc-pentapeptide to free C 55 P, leading to its accumulation in the cytoplasm. During this accumulation, the UDP-MurNAc-pentapeptide is amidated (Figure 10). This amidation of a peptidoglycan precursor was already observed and mediated by AsnB in C. difficile, but only in the presence of vancomycin [START_REF] Ammam | AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin[END_REF]. As vancomycin also targets lipid II, we can hypothesize that the accumulation of UDP-MurNAc-pentapeptide may induce the expression of asnB leading to the amidation of peptidoglycan precursors. UDP-MurNAc-pentapeptide accumulation suggested that the PG synthesis is blocked and explained the essentiality of Lcp activity in C. difficile. In B. subtilis, CWGPs are dispensable for cell viability [START_REF] D'elia | Wall teichoic acid polymers are dispensable for cell viability in Bacillus subtilis[END_REF], but the absence of the three Lcps is lethal [START_REF] Kawai | A widespread family of bacterial cell wall assembly proteins[END_REF].

Similarly, in Mycobacterium tuberculosis, Lcp1, the unique Lcp, was shown to be essential [START_REF] Harrison | Lcp1 Is a Phosphotransferase Responsible for Ligating Arabinogalactan to Peptidoglycan in Mycobacterium tuberculosis[END_REF]. In other bacterial species, this essentiality was not reported, but the absence of Lcp led to defects in growth, morphology, and virulence [START_REF] Chan | Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium[END_REF][START_REF] Schaefer | In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins[END_REF][START_REF] Ballister | Mycobacterium tuberculosis Rv2700 Contributes to Cell Envelope Integrity and Virulence[END_REF]. Our results confirmed the importance of the Lcp proteins in bacterial cell wall organization and their essentiality for bacterial physiology and fitness. Since Lcp proteins are mainly found in Gram-positive and especially in pathogens, they are very good targets for the research of a new class of antibacterial drugs to counteract the emergence of multidrugresistant bacteria.

Materials and methods

Bacterial strains and growth conditions

The strains used and constructed in this study are listed in Table 1. All C. difficile strains of this study are isogenic derivatives of the clinical 630 strain [START_REF] Sebaihia | The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome[END_REF]. C. difficile was grown in a Brain-Heart Infusion medium (BHI, BD Difco) at 37°C in anaerobic conditions (Jacomex, 5% H 2 -5% CO 2 -90% N 2 ). When needed, BHI was supplemented with 1% defibrinated horse blood, thiamphenicol (Th, 7.5 µg.mL -1 ), aztreonam (Az, 16 µg.mL -1 , used to kill parental E. coli during the conjugation process), or erythromycin (Er, 5 µg.mL -1 ). Anhydrotetracycline (ATc) was used to induce the P tet promoter (concentration from 5 to 250 ng.mL -1 ). Growth curves were obtained using a SpectraMax plate reader (Molecular devices). Escherichia coli was grown aerobically in LB medium at 37°C, supplemented when needed with ampicillin (Amp, 100 µg.mL -1 ), chloramphenicol (Cm, 25 µg.mL -1 ), kanamycin (Kn, 40 µg.mL -1 ), spectinomycin (Spec, 100 µg.mL -1 ) or erythromycin (Er, 150 µg.mL -1 ).

Molecular biology

According to the manufacturer's instructions, the plasmid extractions, gel extraction, and PCR purifications were achieved using the Omega E.Z.N.A Plasmid DNA Mini Kit, Gel extraction Kit, and Cycle Pure Kit. PCRs were carried out using high-fidelity Phusion DNA polymerase for gene amplification on genomic DNA and mutant screening of C. difficile. In contrast, the Taq DNA polymerase was used for screening steps in E. coli.

Construction of plasmids

A list of plasmids and primers used in this study can be found in Tables 1 (plasmids), S1, and S2 (primers). The construction of all plasmids is detailed in Text S1. The plasmids used in this study were constructed using either the Gibson assembly protocol from NEB [START_REF] Gibson | Creation of a bacterial cell controlled by a chemically synthesized genome[END_REF] or the Golden Gate assembly from NEB [START_REF] Pryor | Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design[END_REF][START_REF] Pryor | Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design[END_REF] cloning techniques. For Golden Gate assembly, the primers were designed using the NEB Builder® assembly tool.

Mutant strains construction

Plasmids were transferred from E. coli HB101 (pRK24) to C. difficile via heterogramic conjugation (between E. coli and C. difficile), following the previously described protocol [START_REF] Cartman | Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production[END_REF]. The single and double cross-over events were screened based on the pseudosuicide plasmid pMSR following the appropriate protocol described by Peltier [START_REF] Peltier | Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile[END_REF], with some modifications. As we replace the ORFs with a catP gene, a first quick screen for the second crossing-over event is done by restreaking clones on BHI supplemented with a thiamphenicol agar plate. Then, only Th R clones are checked by PCR using appropriate primers (Table S2).

Construction of JMV1, JMV3, and JMV4 strains

The JMV1 strain is an ∆ermB region derivative of the clinical 630 strain. The deletion was made by replacing the complete ermB region [genes CD630_20100 (ermB), CD630_20091, CD630_20090, CD630_20080, CD630_20071, CD630_20070 (ermB)] with a spectinomycin resistance gene. This replacement was made by allelic exchange technique (36) using the pJV8 plasmid.

The single lcp mutants JMV3 and JMV4 are derivatives of the JMV1 strain, where the ORF was replaced with a thiamphenicol resistance gene. The deletion of CD630_27650 (lcpA) and CD630_27660 (lcpB) was made by allelic exchange using respectively deletion plasmids pJV11 and pJV12. The mutants were PCR-verified using the primers couples JV85/JV90, JV86/JV91 for JMV3 mutant, and JV88/JV90, JV87/JV91 for JMV4 strain (Table S2).

Conditional-lethal mutant construction

The insertion of the P tet -lcpB in the erm locus was made using the pJV27 plasmid and the resulting strain JMV2 was PCR-verified using the JV99/JV100 primers. Then, the deletion of both lcp genes was made using the pJV13 plasmid and the use of 100 ng.mL -1 of ATc, giving rise to the conditional-lethal mutant strain JMV6, which can be PCR-checked using primers JV85/JV90 and JV91/JV87 (Table 2).

Beta-glucuronidase assay

The beta-glucuronidase assay was performed as described in Ammam et al., 2020 (38).

Growth and autolysis curves

Growth and autolysis curves were performed using the SpectraMax® plate reader. To ensure anaerobic conditions, the 96-wells plates were filmed with an adhesive film in the anaerobic chamber. The cultures were launched in BHI at an approximate optical density of 0.1 from overnight pre-culture of different strains. Growth and autolysis curves were performed at 37°C. SlpA was purified as described by Bruxelle et al. [START_REF] Bruxelle | Immunogenic properties of the surface layer precursor of Clostridium difficile and vaccination assays in animal models[END_REF] and was submitted to Covalab (France) for guinea pig immunization (four injections with 22.5 µg of the protein per animal). Specificity of the polyclonal antibodies was performed by western blot.

Immunodetection

For the Western blot analyses, the following antibodies were used: anti-SlpA antibodies (guinea pig) diluted at 1:5,000 and anti-Cwp66 antibodies (rabbit) diluted at 1:10,000.

For the dot blot analysis, anti-PSII antibodies (rabbit) diluted 1:10,000 were used.

Antibody binding was revealed with anti-rabbit Immobilon Western Chemiluminescent HRP Substrate (Merck) and revelation was performed on Fusion Fx Imaging System (Vilber Lourmat).

PSII visualization by Super-Resolution Confocal microscope

A 16 h culture was diluted to obtain 10 8 cells.mL -1 thanks to a Kovaslide system, and 20 µL of this diluted culture was deposited on a thin round coverslip. After drying, the slides were stained in TBS-tween BSA 5%, washed, and incubated with the primary antibody (anti-PSII, 1:200) for 1h, with the secondary antibody (StarRED® from Abberior, 1:500) for 1h, and finally with Hoechst (1:2000) to visualize DNA. Washings were performed between each step. Finally, the coverslip was mounted on a slide with mounting medium Abberior Mount Solid® and stored overnight at 4°C before imaging on a STEDYCON super-resolution microscope (Abberior).

PG cytoplasmic precursors extraction and analysis

The protocol described previously by Cremniter et al. [START_REF] Cremniter | Novel mechanism of resistance to glycopeptide antibiotics in Enterococcus faecium[END_REF] was used with some modifications. Bacteria were grown in 500 mL brain heart infusion broth overnight and submitted to ice-cold formic acid (47mL, 1.1M) extraction for 30 min at 4 °C, without prior bacitracin treatment.

To allow comparison of the different strains whose culture optical density was not equal, we calculated the ratio peak area/optical density, presented in the results. The extract was centrifuged (7,000 x g for 15 min at 4 °C) and the supernatant was loaded to a gel filtration column (Sephadex G-25) for desalting. The fraction of elution was lyophilized and resuspended in 10 mL water. 100 µL of this cytoplasmic precursor solution was loaded onto an rpHPLC in a C18 column (Hypersil GOLD aQ; 250 x 4.6 mm; 3 µm, Thermo Scientific) at a flow rate of 1 ml/min. A linear gradient (0 to 20 %) was applied

Statistics

Statistical analyses were conducted using GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, California USA, www.graphpad.com). The p-value is indicated for all comparisons when differences are statistically significant.

Figures legends

The ∆lcpB mutant (JMV4) presents curved, inflated, longer and larger cells than the parental strain JMV1.

A. JMV3 (∆lcpA) and JMV4 (∆lcpB) single mutants were analyzed in optic microscopy and complemented with pMTL84222 (vector), plcpA (plasmid carrying lcpA expressed with its own promoter) or plcpB (plasmid carrying lcpB expressed with its own promoter).

Scale bar represents 20µm. B. The percentage of abnormal (curved, thick, or inflated) cells among total cells was calculated by measuring more than 100 cells for each strain.

C. Cell length of the parental strain JMV1, JMV3 and JMV4 complemented with the vector (pMTL84222), plcpA or plcpB. D. Cell width of the parental strain JMV1, JMV3 and JMV4 complemented with the vector (pMTL84222), plcpA or plcpB. For B, C and D, the number above each group data represents the number of cells counted. **** means student t-test p-value is< 0.0001.

Figure 2

Both lcpA and lcpB have a constitutive expression GusA activity measured for lcpA (A) and lcpB (B) promoters GusA activity (blue curve) was measured in Miller Units and growth (red curve) was followed by measuring the optical density at 600 nm (OD 600nm ) for 8 hours.

Figure 3

Both ∆lcpA and ∆lcpB exhibit an altered PSII layer at the surface.

Immunofluorescence assay of JMV1, JMV3 and JMV4 strains, was observed using super-resolution microscope. Bacteria were stained for DNA (DAPI, blue) and PSII (anti-PSII, green). The merged picture shows both localizations simultaneously (scale bar represents 20 µm). The panel shows a magnify part of the picture (scale bar represents 2 µm).

Figure 4 The lcp conditional-lethal mutant (JMV6) is not able to grow without ATc induction of the P tet -lcpB copy.

A. JMV1 and JMV2 grown in liquid culture were diluted and plated on BHI agar petri dishes. JMV6 grown in liquid culture in the presence of 10 (JMV6 (ATC 10)) or 50 ng.mL -1 (JMV6 (ATc 50)) of ATc was diluted and plated on BHI agar petri dishes. The control strain, JMV6 harboring the plasmid plcpA grown in liquid culture in the presence of 50 ng.mL -1 of ATc (JMV6 + plcpA (ATc 50)) was diluted and plated on BHI agar petri dishes. Petri dishes contained ATc from 0 to 250 ng.mL -1 in the BHI agar medium. B. JMV1, JMV2, JMV6 and JMV6 + plcpA were grown in the presence of 50 ng.mL -1 of ATc and then, the growth was measured for 20h (1200 minutes) without ATc (JMV1, JMV6 + plcpA), in the presence of 10 ng.mL -1 of ATc (JMV6 (ATc 10)) or in the presence of 50 ng.mL -1 of ATc (JMV2 (ATc 50), JMV6 (ATc 50), JMV6 + plcpA (ATc 50)). The graph represents the mean of 3 independent experiments.

Figure 5

In the presence of 10 ng.mL -1 of ATc, the lcp conditional-lethal mutant (JMV6) loses its rod shape.

A. JMV1, JMV6 + plcpA, JMV6 + plcpB, JMV2 in the presence of 50 ng.mL -1 (JMV2 (ATc 50)) of ATc and JMV6 in the presence of 10 (JMV6 (ATc 10)) or 50 ng.mL -1 (JMV6 (ATc 50) of ATc were observed in optic microscopy. The scale bar represents 20 µm. B and C. Cell length (B) and cell width (C) of bacteria from each strain observed in A.

were measured. The number above each group data represents the number of cells counted. **** means student t-test p-value is< 0.0001.

Figure 6

In the presence of 10 ng.mL -1 of ATc , the lcp conditional-lethal mutant (JMV6) loses its rod shape but PSII is still detected at the surface.

Immunofluorescence assay of JMV1, JMV2 in the presence of 50 ng.mL -1 (JMV2 (ATc50)), JMV6 in the presence of 10 ng.mL -1 (JMV6 (ATc10)), JMV6 in the presence of 50 ng.mL -1 (JMV6 (ATc50)) and JMV6 + plcpA in the presence of 50 ng.mL -1 (JMV6 + plcpA (ATc50)) strains was observed using super-resolution microscope. Bacteria were stained for DNA (DAPI, blue) and PSII (anti-PSII, green). The merged picture shows both localizations simultaneously (scale bar represents 20µm). The panel shows a magnify part of the picture (scale bar represents 2µm). Figure 7 PSII is released into the supernatant of the JMV6 strain in the presence of 10 ng.mL -1 of ATc Dot blot analysis using specific antibodies targeting PSII was performed on the bacterial surface content (pellet) and supernatant content (culture supernatant) from JMV1, JMV2 JMV3, JMV4, JMV6 grown in the presence of 10 ng.mL -1 of Atc (JMV6 (Atc 10)), JMV6 grown in the presence of 50 ng.mL -1 of Atc (JMV6 (Atc 50)) and JMV6 plcpA grown in the presence of 50 ng.mL -1 of Atc (JMV6 + plcpA (Atc 50)). Each content was diluted up to 1:64. PG-PSII is used as positive control and PG as a negative control. Characterization of surface (A, C, E) and supernatant (B, D, F) protein profiles from JMV1, JMV2, JMV6 and JMV6 + plcpA grown in the absence of Atc (-) or in the presence of 10 ng.mL -1 of Atc [START_REF] Meireles | Listeria monocytogenes Wall Teichoic Acid Glycosylation Promotes Surface Anchoring of Virulence Factors, Resistance to Antimicrobial Peptides, and Decreased Susceptibility to Antibiotics[END_REF] or in the presence of 50 ng.mL -1 of Atc [START_REF] Cartman | Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production[END_REF].

Coomassie blue staining (A, B), anti-Cwp66 Western blots (C, D) and anti-SlpA Western blots (E, F) were performed. The protein ladder is graduated in kg Dalton (kDa). MW: molecular weight.

PSII anchoring mutants present an autolysis phenotype

Autolysis of JMV1, JMV3 and JMV4, harboring the empty plasmid pMTL84222 (+ vector), the plcpA plasmid (+ plcpA) or the plcpB plasmid (+ plcpB) were measured and presented in A. Autolysis of JMV1, JMV2 grown in the presence of 50 ng.mL -1 of ATc [START_REF] Cartman | Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production[END_REF], JMV6 + plcpA, JMV6 grown in the presence of 10 ng.mL -1 of ATc (JMV6 (ATc10)), JMV6 grown in the presence of 50 ng.mL -1 of ATc (JMV6 (ATc50)) and JMV6 + plcpA grown in the presence of 50 ng.mL -1 of ATc (JMV6 + plcpA (ATc50)), were measured and presented in B. The optical density was measured for 3 hours (180 minutes) and the result is presented as a cell survival percentage. The graph represents the mean of 3 independent experiments. Figure 10 PG cytoplasmic precursors accumulate when PSII anchoring is impaired A, B and C. Purification and quantification of UDP-MurNAc-pentapeptide from JMV1 (A), JMV6 grown in the presence of 10 ng.mL -1 of ATc (JMV6 (ATc10)) (B), JMV6 + plcpA grown in the presence of 50 ng.mL -1 of ATc (JMV6 + plcpA (ATc50)) (C) was performed.

D.

A table represents each peak area and area/optical density ratio. In addition, the observed and calculated monoisotopic masses obtained after mass spectrometry analysis are presented in the two last columns. mAU = milli Arbitrary Unit, Da = Dalton

Supplemental figures legends

Figure S1

Graphic map of the pJV10 plasmid used to construct deletion plasmids of the lcp On this graphic map of the pJV10 plasmid, created by Serial Cloner, the spectinomycin resistance gene flanked by BsaI sites to allow Golden Gate assembly, an erythromycin resistance gene, and the P tet -CD2517 (Toxin) from the pMSR to facilitate counterselection during the allelic exchange are shown.

Figure S2

The ∆lcpB strain presents an altered growth Growth curve of single mutant strains of lcpA (JMV3) and lcpB (JMV4), harboring either the pMTL84222, or the plcpA or plcpB plasmid. The growth was observed in BHI medium for 17 hours (1020 minutes). The graph represents the mean of 3 independent experiments.

Figure S3

The ∆lcpB mutant (JMV4) is thicker, curved, or inflated in liquid culture.

These panels present additional pictures of the JMV4 strain observed in optic microscopy. The scale bar represents 20 µm.

Figure S4

The single lcp mutants JMV3 and JMV4 exhibit a normal S-layer content This Coomassie staining of Cwp protein extractions shows that the Cwp content of the S-layer of JMV1, JMV3 and JMV4 harboring either pMTL84222 (vector), plcpA or plcpB plasmid. The protein ladder is graduated in kg Dalton (kDa). MW: molecular weight.

Figure S5

The PSII was obtained and the absence of contamination with LTA was confirmed by NMR.

1 H (A) and 31 P (B) NMR spectra of the PSII extracted from culture pellets of the 630 strain. Two samples were sent for analysis, named PSII 3.2 and PSII 2.6. Both were confirmed to contain PSII. The chemical shift is measured in part-per-million (ppm).

Figure S6

The immunization led to antibodies production and these antibodies showed good specificity for the PSII This dot blot assay shows that the antibodies produced by the rabbits recognize well the RMN-verified PSII (A) of C. difficile and the PG linked PSII (PG-PSII) (A and C) and do not cross-react with peptidoglycan (A and C) or lipoteichoic acid (LTA) (C) of C. difficile.

Moreover, different samples at different stages of the purification process were tested (B). Briefly, PSII purification protocol was performed as followed (white boxes, steps of PSII purification, yellow boxes, potential contaminant molecules) : 1 litter of C. difficile culture was pelleted. Pellet was washed in PBS and boiled in water for 30 minutes. After centrifugation, pellet (a) was tested to know whether some PSII were not recovered. The supernatant was further used for purification and a TCA precipitation was performed.

After centrifugation, pellet (b) was tested to know whether some PSII was not recovered; it was resuspended in water and centrifuged again, giving pellet (c). The supernatant (d) was dialyzed and applied on fPLC. PSII was recovered, dosage was performed by phenol sulfuric method and PSII was analyzed by NMR.

The ∆lcpB mutant (JMV4) is thicker, curved, or inflated in liquid culture.

These panels present additional pictures of the JMV4 strain observed in optic microscopy. The scale bar represents 20 µm.

The PSII was obtained and the absence of contamination with LTA was confirmed by NMR.

The anchoring of PSII is essential for C. difficile growth

The conditional-lethal mutant pre-cultured overnight in liquid BHI in the presence of 50 ng.mL -1 ATc is not able to grow on a BHI plate without ATc but grows correctly in the presence of ATc at 50 ng.mL -1 .

  between 13 and 33 min at 25 °C (buffer A, 50 mM ammonium formate pH 4.4; buffer B, 100 % Methanol). Absorbance was monitored at 262 nm and the peak corresponding to the major cytoplasmic precursor was collected, lyophilized, resuspended in 20 µL of water. Ten µL were analyzed by mass spectrometry on a Bruker Daltonics maXis highresolution mass spectrometer (Bremen, Germany) operating in the positive mode (Analytical platform of the Muséum National d'Histoire Naturelle, Paris, France). Mass spectral data were explored using Bruker Compass DataAnalysis 4.3.

Figure 8 PSII

 8 Figure 8 PSII anchoring impairment is associated with Cwp proteins released in the culture supernatant.

Figure 9

 9 Figure 9

Figure S7 A

 S7 Figure S7A new strategy designed to construct a conditional-lethal mutant in C. difficile Schematic representation of the strategy used to create a conditional-lethal mutant of C. difficile lcpA and lcpB genes. The strategy consists in three major steps: first the insertion of an inducible copy of lcpB in the erm locus of the chromosome (under control of P tet ), then the deletion of both lcpA and lcpB by replacing the ORFs with a catP gene, and finally the control of the l expression of lcpB thanks to ATc induction.

FiguresFigure S1

 S1 Figures

Figure S2 The

 S2 Figure S2

Figure S4 The

 S4 Figure S4

1 H

 1 (A) and 31 P (B) NMR spectra of the PSII extracted from culture pellets of the 630 strain. Two samples were sent for analysis, named PSII 3.2 and PSII 2.6. Both were confirmed to contain PSII. The chemical shift is measured in part-per-million (ppm).

Figure S6 The

 S6 Figure S6

Figure S7 A

 S7 Figure S7

Tables : Table 1 .

 :1 Bacterial strains and plasmids used in this study.

		pAT28	Mobilizable shuttle plasmid, Spc R	Trieu-Cuot et
				al. 1990 (57)
		pRPF185	P tet -gusA Tm R, expression and cloning vector	Fagan et al.
				2011 (58)
		Name pMTL-83151 pMTL-84151 pMTL-84222	Genotype or primer sequence Bacterial strains Cm R cloning vector, pCB102 replicative origin Cm R cloning vector, pCD6 replicative origin Erm R cloning vector, pCD6 replicative origin	Source or reference Heap et al. 2009 (59)
		pJV4 Escherichia coli TG1 pJV5 HB101 pRK24 pJV6	pBLUNTΩaadA, spectinomycin resistance gene E. coli k12 (F', tra D36, lacIq, ∆lacZ, MIS, pro A+B+/SupE, ∆(hdsM-mcrB)) flanked by BsaI sites, Kn R pMSR derivative, used to construct pJV8, Cm R and E. coli (pRK24) (F -∆(gpt-proA) 62 Leu B6 gln V44 ara-14 galK2 lacY1 ∆(mcrC-mrr) rps L20 (srt r ) xyl-5 Sp R pMTL-83151ΔcatPΩermB, Erm R	This work Laboratory This work stock Laboratory stock This work
		Clostridioides difficile pJV7	flanked by BsaI sites, Cm R and Sp R mlt-1 rec A13, pRK24 pMSRΩaadA, spectinomycin resistance gene	This work
		630 pJV8 630∆erm pJV10	Clinical strain, Erm R pMSR derivative, plasmid used for Erm locus deletion, Cm R, and Sp R Derivative of 630 strain, Erm S pJV7ΔcatPΩermB, Erm R and Sp R	This work Sebaihia et al. 2006 (46) Hussain et al. This work
		JMV1 pJV11	Derivative of 630 strain, Erm S , ∆(CD630_20100, plasmid, Erm R pJV10 derivative, plasmid used for lcpA deletion	This work 2005 (37) This work
		pJV12	CD630_20071, CD630_20070) plasmid, Erm R CD630_20091, CD630_20090, CD630_20080, pJV10 derivative, plasmid used for lcpB deletion	This work
		JMV2 (630 P tet -lcpB) pJV13	Derivative of 630 strain, Erm S , ∆(CD630_20100, CD630_20091, CD630_20090, CD630_20080, deletion, Erm R pJV10 derivative, plasmid used for lcp region	This work This work
		pTC131 JMV3 (∆lcpA) JMV4 pMEZ5 JMV5 (∆lcpB P tet -plcpA (pMEZ12) pJV20 lcpB) JMV6 (∆lcpA plcpB (pJV21) pJV27 ∆lcpB P tet -lcpB) pMDR1	pMTL-84151ΩaadA, spectinomycin resistance CD630_20071, CD630_20070)::P tet -lcpB JMV1 ∆lcpA gene flanked by BsaI sites, Cm R and Sp R JMV1 ∆lcpB pTC131ΔcatPΩermB, Erm R and Sp R JMV2 ∆lcpB pMEZ-5ΩP lcpA -lcpA, Erm R pTC131ΩP lcpB -lcpB, Cm R JMV2 ∆lcpA ∆lcpB pMTL-84222ΩP lcpB -lcpB, Erm R pJV8ΩP tet -lcpB, Cm R Plasmids and vectors pTC131ΩaphAΩgusA, Cm R, and Kn R	This work This work This work This work This work This work This work This work This work This work This work
		pMSR pMDR2	Circular cloning vector, 5624 nucleotides, catP, pMDR1ΔaphAΩaadA, Cm R and Sp R	Gift from J. This work
		pMDR5 pMDR8	pMDR2ΔaadAΩP lcpB , Cm R αlacZ, P tet -CD2517 (toxin), pseudo-suicide plasmid, Cm R pMDR2ΔaadAΩP lcpA , Cm R	Peltier (36) This work This work
	868	pBLUNT	Linear cloning vector from Invitrogen, Kn R	Invitrogen

Acknowledgments

We thank Johann Peltier for providing the pMSR plasmid for allelic exchange in C. difficile, R.P. Fagan for the gift of pRPF185, Afi Akofa Diane Sapa for the purification of SlpA, Assilina Parfut, Marie-Emeline Zielinski and Mathieu Rodriguez for plasmids constructions. We also thank Valerie Nicolas of the platform MIPSIT of Paris Saclay University for immunofluorescence imaging. Finally, we want to thank Frederic Eghiaian from Abberior company for his help for super-resolution microscopy experiments.

Footnotes

This paper contains supplementary materials.

Funding

This work was funded by a PhD grant of the Ministère de l'Education Nationale, de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI) to Jeanne Malet-Villemagne and a National Institute of Allergy and Infectious Diseases (R56AI045626) grant to Yucheng Liang.

Cwp proteins and supernatant proteins extractions

Cwp proteins were isolated from intact C. difficile bacteria using low-pH glycine as described previously by Fagan et al. [START_REF] Fagan | Dissecting the cell surface[END_REF]. The optical density was systematically adjusted to 1 for all strains before protein extraction. Supernatant proteins fraction was obtained by harvesting bacteria (20,000 x g, 15 min, 4 °C) from overnight cultures previously adjusted to an optical density (600 nm) of 1, and then precipitated with trichloroacetic acid 10% (on ice, 4 hours). The pellet is finally resuspended in Tris 50 mM pH 7.4.

Preparation of antigens and antibodies against PSII and SlpA

Surface polysaccharide II was isolated using the protocol of Cox [START_REF] Cox | Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection[END_REF]. The detection of glycopolymers in fPLC fractions was accomplished by the phenol-sulfuric assay [START_REF] Michel | Colorimetric Method for Determination of Sugars and Related Substances[END_REF].

The fractions of interest were freeze-dried and analyzed by 1 H and 31 P NMR (ENS Lyon) to confirm the PSII purification. Purified NMR-confirmed PSII was then conjugated to bovine serum albumin (BSA). The coupling reaction proceeded according to the protocol described by Romano [START_REF] Romano | Recombinant Clostridium difficile toxin fragments as carrier protein for PSII surface polysaccharide preserve their neutralizing activity[END_REF], with cyanoborohydride (NaBH 3 CN) as a coupling agent. The resulting glycoconjugate antigen (PSII-BSA) was submitted to Covalab (France) for rabbit immunization (four injections with 50 µg of the glycoconjugate per animal). Specificity of the purified PSII was confirmed by dot blot and NMR-confirmed, peptidoglycan (PG), and PG-PSII extracts.

Figure S8

The anchoring of PSII is essential for C. difficile growth

The conditional-lethal mutant pre-cultured overnight in liquid BHI in the presence of 50 ng.mL -1 ATc is not able to grow on a BHI plate without ATc but grows correctly in the presence of ATc at 50 ng.mL -

Texts

Text S1: construction of plasmids for the study

The plasmids used in this study were constructed using either the Gibson assembly protocol from NEB [START_REF] Chan | Staphylococcus aureus mutants lacking the LytR-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium[END_REF] or the Golden Gate assembly from NEB (24, 25) cloning technique. For Golden Gate assembly, the primers were designed using the NEB Builder® assembly tool.

Construction of deletion plasmid for the ermB locus

pJV4: the spectinomycin cassette was amplified from pAT28 and flanked with BsaI sites using JV54/JV55 primers. The PCR product was inserted in the pBLUNT cloning vector by blunt-end DNA cloning to give pJV4.

pJV5: the spectinomycin cassette flanked with BsaI sites was amplified from pAT28 using JV50/JV51 primers, and the downstream region of ermB locus was amplified from genomic DNA of 630 strain using JV52/53 primers. Both PCR products were assembled using the Gibson assembly protocol (NEB Biolabs) to give pJV5.

pJV7: the spectinomycin cassette flanked with BsaI sites was extracted from pJV4 using restriction digestion with XhoI and BamHI. The pMSR was opened by restriction digestion with XhoI and BamHI. The spectinomycin cassette was then cloned into the linearized pMSR plasmid by a classical ligation process.

pJV8: the upstream region of the ermB locus was amplified from genomic DNA of 630 strain using JV48/JV49 primers, and the spectinomycin + downstream region of ermB locus fragment was amplified from pJV5 using JV50/JV53 primers, and the pMSR plasmid was amplified using JV46/JV47 primers. The three PCR products were assembled using the Gibson assembly protocol (NEB Biolabs) to give pJV8.

Constructing deletion plasmids for lcpA, lcpB and the conditional-lethal deletion of both lcp pJV6: the ermB cassette was amplified from pMTL84222 using TC289/TC290 primers, and pMTL83151 was amplified using TC287/TC288 primers. Both PCR products were assembled using the Gibson assembly protocol (NEB Biolabs) to give pJV6. pJV10: this plasmid results from the subcloning of pJV7 (fragment with the spectinomycin resistance cassette flanked by BsaI sites and Ptet-CD2517 toxin) into pJV6 (Erm R ) using the restriction enzymes SacII and XhoI.

pJV11: the upstream and downstream regions of lcpA were amplified from genomic DNA of 630 strain using respectively JV58/59 and JV62/JV63 primers. The catP cassette was amplified from the pMSR plasmid using JV60/JV61 primers. The three PCR products were inserted in the pJV10 using the Golden Gate assembly protocol (NEB Biolabs) to give pJV11.

pJV12: the upstream and downstreamregions of lcpB were amplified from genomic DNA of 630 strain using respectively JV64/65 and JV68/JV69 primers. The catP cassette was amplified from the pMSR plasmid using JV66/JV67 primers. The three PCR products were inserted in the pJV10 using the Golden Gate assembly protocol (NEB Biolabs) to give pJV11.

pJV13: the upstream region of lcpA and the downstream region of lcpB were amplified from genomic DNA of 630 strain using respectively JV58/JV70 and JV73/JV69 primers. The catP cassette was amplified with its promoter from the pMSR plasmid using JV71/JV72 primers.

The three PCR products were inserted in the pJV10 using the Golden Gate assembly protocol (NEB Biolabs) to give pJV11.

Constructing complementation plasmids for lcpA and lcpB pTC131: pJV4 was digested using BamHI/XhoI restriction enzymes, and the DNA fragment Spec of approximately 1kb was ligated pMTL84151 previously digested by BamHI/XhoI restriction enzymes.

pMEZ5: The ermB cassette from pMTL-84222 was amplified using TC383/TC384 primers, and pTC131 was amplified using TC381 and TC382 primers. Both PCR products were assembled using the Gibson assembly protocol (NEB Biolabs) to give pMEZ5.

pMEZ12: PlcpA-lcpA was amplified from genomic DNA of 630 strain using TC403/TC404 primers and inserted in pMEZ5 using the Golden Gate assembly protocol (NEB Biolabs) to give pMEZ12. pJV20: PlcpB-lcpB was amplified from genomic DNA of 630 strain using JV101/JV102 and inserted in pTC131 using the Golden Gate assembly protocol (NEB Biolabs) to give pJV20.

pJV21: pJV20 was digested using StuI and KpnI restriction enzymes, and the DNA fragment of approximately 1,5kb was ligated with pMTL84222 (Erm R ) previously digested by StuI and KpnI restriction enzymes.

pJV27: the Ptet was amplified from pRPF185 using JV136/JV137 primers, and the lcpB was amplified from genomic DNA of 630 strain using JV138/JV139 primers. Both PCR products were inserted in pJV8 using the Gibson assembly protocol (NEB Biolabs) to give pJV27.

Cloning gusA reporter plasmids pMDR1: Kanamycin cassette flanked with BsmBI restriction enzyme sites was amplified from pJV4 using TC393/TC394 primers. gusA was amplified from pRPF185 using TC395/TC396 primers. Both PCR DNA fragments were inserted into pTC131 using the Golden Gate assembly protocol to give pMDR1. pMDR2: Spectinomycin cassette was amplified from pAT28 using TC397/TC398 and inserted into pMDR1 to give pMDR2.

pMDR8: PlcpA was amplified from genomic DNA of 630 strain using TC409/TC410 and inserted into pMDR2 to give pMDR8. PlcpB was amplified from genomic DNA of 630 strain using TC411/TC412 and inserted into pMDR2 to give pMDR5.