Héloise Coullon

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

Clostridioides difficile peptidoglycan modifications

The cortex and peptidoglycan of Clostridioides difficile have been poorly investigated. This last decade, the interest increased because these two structures are highly modified and these modifications may be involved in antimicrobial resistance. For example, C. difficile peptidoglycan deacetylation was recently reported to be involved in lysozyme resistance. Modifications may also be important for spore cortex synthesis or spore germination, which is essential in C. difficile pathogenesis. As such, the enzymes responsible for modifications of the peptidoglycan and/or cortex could be new drug target candidates or used as anti-C. difficile agents, as seen for the CD11 autolysin. In this review, we focused on C. difficile peptidoglycan and cortex and compared their structures with those of other well studied bacteria.

Introduction

Peptidoglycan is specific and unique to bacteria [START_REF] Pazos | Peptidoglycan[END_REF]. It surrounds bacteria and is necessary for bacterial growth. In C. difficile, this structure is found as a part of the vegetative cell wall but also in the cortex, which is a peptidoglycan-like structure found in the spore [START_REF] Popham | Spore Peptidoglycan[END_REF]. Many peptidoglycan modifications are reported and some may be crucial for the bacterial survival and way of life [START_REF] Vollmer | Peptidoglycan structure and architecture[END_REF]. In this review, we will focus on the C. difficile peptidoglycan and cortex structures and their modifications in comparison with those of other well-studied bacteria.

Peptidoglycan structure

The amino acids found in the pentapeptide stem of C. difficile peptidoglycan are similar to that of gram negative or Bacillus genus bacteria, including D-Alanine, L-Alanine, D-Glutamate and meso diaminopimelic acid (A2pm) [START_REF] Peltier | Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links[END_REF] (Figure 1A). In addition to these amino acids, several new amino acids such as phenylalanine, lysine, valine or modified alanine were identified in low amounts in the muropeptidesof the 630erm strain using LC-high-resolution mass spectrometry [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF]. Aside from the composition of the pentapeptide stem, the bacterial peptidoglycan of C. difficile has several highly unique characteristics. Amongst these characteristics, it contains a majority of A2pm 3 → A2pm 3 crosslinks (2,6 diaminopimelic acid linked in position 3 of the peptide of the peptidoglycan) instead of D-Ala→ A2pm 3 cross-links found in the majority of bacteria (Figure 1A) [6]. Through a combination of genetic knock-out studies and characterization of recombinant proteins, three proteins have been shown to be L,D-transpeptidases with partially redundant functions leading to the formation of A2pm 3 → A2pm 3 cross-links [START_REF] Sütterlin | Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams[END_REF]. However, discrepancies between these proteins could hint at differences in localization, substrate specificity, antibiotic sensitivity or even essentiality. Indeed, inactivation of ldtcd1 and ldtcd2 lead to a decrease in the abundance of A2pm 3 → A2pm 3 cross-links in peptidoglycan. In contrast, no mutant could be generated for ldtcd3, suggesting that ldtcd3 is essential [START_REF] Sütterlin | Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams[END_REF]. Recombinant proteins of Ldtcd1 and Ldtcd2 were found to be the targets of carbapenems, while this was not seen for the recombinant protein of Ldtcd3 [START_REF] Sütterlin | Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams[END_REF]. Incubation of recombinant proteins with disaccharide-tetrapeptide revealed that Ldtcd2 and Ldtcd3 display L,D-transpeptidase and L,Dcarboxypeptidase activities. In contrast, the Ldtcd1 recombinant protein displayed only L,D carboxypeptidase activity in these experimental conditions [START_REF] Sütterlin | Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams[END_REF]. Finally, it is important to note that the L,D-transpeptidase encoded by ldtcd2, Cpw22, has recently been studied and shown to be involved in cell wall integrity and viability in the R20291 strain [START_REF] Zhu | Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile[END_REF].

Peptidoglycan deacetylation

The second particularity of the C. difficile peptidoglycan is the high N-deacetylation of the glucosamine (93 to 97.5% according to the studies) [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF][START_REF] Ho | Clostridium difficile extracytoplasmic function σ factor σV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF][START_REF] Kaus | Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases[END_REF]. Among 12 potential N-deacetylases encoded in the genome of C. difficile, at least two are involved in the peptidoglycan N-deacetylation and lysozyme resistance. These enzymes, PdaV and PgdA, act in synergy and are regulated by the extracytoplasmic function  V (sigma factor V) [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF][START_REF] Ho | Clostridium difficile extracytoplasmic function σ factor σV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF][START_REF] Kaus | Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases[END_REF]. pdaV expression is fully dependent on  V whereas pgdA is still expressed in a csfV mutant (csfV encodes  V ) suggesting a difference of regulation for both genes. Each individual mutant, pgdA or csfV, is slightly more sensitive to lysozyme than the parental strain (4-fold in the 630erm and 2-fold and 8-fold respectively in the R20291 [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF][START_REF] Kaus | Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases[END_REF]). In contrast, the double mutant is more than 130-fold more sensitive to lysozyme in the 630erm strain and 1000-fold in the R20291 strain [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF][START_REF] Ho | Clostridium difficile extracytoplasmic function σ factor σV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF]. In other pathogenic bacteria such as Streptococcus pneumoniae or Listeria monocytogenes, the absence of peptidoglycan N-deacetylation altered the virulence [START_REF] Boneca | A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system[END_REF][START_REF] Vollmer | Peptidoglycan N-acetylglucosamine deacetylase, a putative virulence factor in Streptococcus pneumoniae[END_REF]. In contrast, in vivo virulence studies done in C. difficile showed that although the intestinal colonization is lower for the double mutant ΔpgdA csfV compared to the parental strain, the double mutant is more virulent than the parental strain [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF]. A possible explanation for this observation, that need to be further investigated, is that the increased lysozyme sensitivity of the double mutant, shown in vitro, could lead to a stronger bacterial lysis during in vivo infection. This could facilitate the release of toxins TcdA and TcdB [START_REF] El Meouche | Toxin release mediated by the novel autolysin Cwp19 in Clostridium difficile[END_REF][START_REF] Govind | Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE[END_REF], ultimately leading to a higher mortality of the host.

Peptidoglycan modifications in the presence of vancomycin

Peptidoglycan modifications may be also involved in antibiotic resistance [START_REF] Rajagopal | Envelope Structures of Gram-Positive Bacteria[END_REF]. Indeed, bacterial resistance to vancomycin has been linked to the presence of a van gene cluster [16]. While 74% of C. difficile strains possess a vanGCd gene cluster [17,[START_REF] Peltier | Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile[END_REF], only a few clinical strains have been found to harbor a vancomycin resistance [START_REF] Shen | Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates[END_REF]. The vanGCd gene cluster is composed of two operons: a regulation operon (vanR and vanS) and a resistance operon (vanG, vanXY and vanT). The regulation operon is constitutively expressed, whereas the resistance operon is induced by the presence of vancomycin through the two-component system VanS-VanR. In the presence of vancomycin, the peptidoglycan precursors are modified and close to 40% of them are UDP-MurNac-pentapeptide[D-Ser] instead of the natural peptidoglycan precursor UDP-MurNac-pentapeptide[D-Ala] [17]. This low rate of modified precursors may lead to the slight change in vancomycin susceptibility between the vanG or vanR mutants (MIC 0.75 mg L -1 ) and the parental strain (MIC 1.5 mg L -1 ). The lack of resistance and low rate of modified precursor UDP-MurNac-pentapeptide[D-Ser] may be explained by the absence of D,Dcarboxypeptidase activity of VanXY and the fact that MurF has a preferential use of lipid II ending with D-Ala-D-Ala instead of D-Ala-D-Ser [17]. In addition, in the presence of vancomycin, Ammam et al. reported amidation of a subset of peptidoglycan precursors [START_REF] Ammam | AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin[END_REF]. This modification was only found in the presence of vancomycin and was independent from the VanS-VanR two-component system. Intriguingly, when the asparagine synthetase asnB was overexpressed, more than 90% of the peptidoglycan precursors were amidated, but the growth or morphology of the strain was similar to that of the parental strain [START_REF] Ammam | AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin[END_REF]. In contrast, the overexpression of asnB led to a decrease of the vancomycin MIC from 2-3 to 1-1.5 mg L -1 [START_REF] Ammam | AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin[END_REF]. Finally, Shen et al. recently showed that a weak expression of the resistance operon is involved in the lack of vancomycin resistance [START_REF] Shen | Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates[END_REF]. Indeed, punctual mutations in vanS or vanR may increase the expression of the resistance operon, leading to an increase of the MIC from 4 to 16 mg L -1 . In addition, Pu et al. identified a transferable plasmid (pX18-498) that is found in clinical strains associated with vancomycin treatment failure in CDI patients [START_REF] Pu | Plasmid Acquisition Alters Vancomycin Susceptibility in Clostridioides difficile[END_REF]. This plasmid may result in decreased susceptibility to vancomycin. The combination of such a plasmid with mutations in vanR and/or vanS may result in increasing rates of vancomycin resistance in C. difficile strains.

Peptidoglycan autolysins

Autolysins are enzymes that maturate the peptidoglycan and are be involved in toxins release [START_REF] Elahi | The Human Gut Microbe Bacteroides thetaiotaomicron Suppresses Toxin Release from Clostridium difficile by Inhibiting Autolysis[END_REF]. Several were recently predicted in C. difficile [START_REF] Mitchell | Building blocks and blueprints for bacterial autolysins[END_REF]. Among them, the N-acetylglucosaminidase Acd was studied using B. subtilis peptidoglycan (highly acetylated on its glucosamine) [START_REF] Dhalluin | Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity[END_REF]. It would be worth to know whether this enzyme is also able to act on deacetylated muropeptides since C. difficile peptidoglycan is highly deacetylated. A second autolysin, Cwp19, is a lytic transglycosidase that cleaves the peptidoglycan between the N-acetyl muramic acid and the N-acetyl glucosamine [START_REF] El Meouche | Toxin release mediated by the novel autolysin Cwp19 in Clostridium difficile[END_REF][START_REF] Bradshaw | The molecular structure of the glycoside hydrolase domain of Cwp19 from Clostridium difficile[END_REF][START_REF] Wydau-Dematteis | Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile[END_REF]. Interestingly, it was shown that the toxins, TcdA and TcdB, were released due to the autolysis induced by Cwp19, in a process that appears media specific [START_REF] Wydau-Dematteis | Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile[END_REF]. This autolysin is expressed during the active phase of growth (mainly up to 24h of growth), but its activity is detected during the late stationary phase of growth (from 24h). The hypothesis behind this apparent inconsistency is that a secondary polymer like the PSII or the LTA could modulate its autolytic activity. However, CWP proteins, including Cwp19, are suggested to be placed at the top of the PSII [START_REF] Usenik | The CWB2 Cell Wall-Anchoring Module Is Revealed by the Crystal Structures of the Clostridium difficile Cell Wall Proteins Cwp8 and Cwp6[END_REF] and therefore should be resistant to the shielding-like activity proposed. Two N-acetylmuramoyl-L-alanine amidases, CD11 and CDG, are produced by C. difficile prophages and were identified as clinical candidates to target C. difficile infection [START_REF] Wu | Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile[END_REF]. These enzymes may have a role in the peptidoglycan maturation. However, it was later shown that CD11 is able to kill C. difficile bacteria in PBS, but is mostly inactivated by the wall teichoic acids [START_REF] Wu | Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile[END_REF]. CwlA is a peptidoglycan hydrolase described in the 630erm (CD630_11350) and in the R20291 (Cwl0971) strains [START_REF] Garcia-Garcia | Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile[END_REF][START_REF] Zhu | Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291[END_REF]. This endopeptidase has three SH3_3 domains (also named SH3_b [START_REF] Desvaux | Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display[END_REF]) and a NLPC/60 super family catalytic domain. CwlA cleaves the stem peptide of the peptidoglycan between the γ-D-Glu and mDAP. CwlA is predominantly found at the septum, and in its absence, C. difficile has a cell separation defect. In the R20291 strain, cwlA was found to be mainly expressed in the exponential phase of growth and in a lesser extent during the stationary phase. Recent work has shown that CwlA is in fact controlled by the Serine/Threonine kinases PrkC and CD630_21480 [START_REF] Garcia-Garcia | Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile[END_REF][START_REF] Cuenot | The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile[END_REF]. The proposed model is that PrkC is able to phosphorylate CwlA, which leads to cytoplasmic localization of CwlA. In contrast, unphosphorylated CwlA is exported in the cell wall where it is able to cleave the peptidoglycan [START_REF] Garcia-Garcia | Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile[END_REF]. This is a particular regulation that was not reported previously.

Spore cortex

The composition and structure of C. difficile spore cortex has recently been extensively characterized [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF] (Figure 1B). It consists in muropeptides that are predominantly found as monomers (approximately 90% of muropeptides) and a minority found as dimers, leading to a very low crosslinking index (< 5%). Muramic-δ-lactams were detected on 24% of the muropeptides, in contrast with B. subtilis [START_REF] Popham | Analysis of the peptidoglycan structure of Bacillus subtilis endospores[END_REF] or C. perfringens [START_REF] Orsburn | Factors contributing to heat resistance of Clostridium perfringens endospores[END_REF] where 50% of muropeptides contained muramic-δ-lactams. Unlike species such as B. subtilis where a single PdaA N-deacetylase is involved in muramic-δ-lactam synthesis [START_REF] Gilmore | Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan[END_REF], two N-deacetylases were found to be involved in the muramic-δ-lactam synthesis in C. difficile: PdaA1 and PdaA2 (CD630_14300 and CD630_27190 respectively) [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF]. These two enzymes are forming the δ-lactams after the processing of muramic acid and its stem peptide by GerS and CwlD [START_REF] Diaz | Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination. mSphere 2018, 3. This publication analyzed the synthesis of δ-lactams in the spore cortex of C. difficile by GerS, CwlD and PdaA 38[END_REF]38]. A mutant of pdaA1 and pdaA2 exhibits a decreased sporulation, a decreased heat resistance, delayed virulence in a hamster infection model, and an altered germination [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF]. In contrast, B. subtilis cannot germinate if the muropeptides lack muramic-δ-lactams [START_REF] Popham | Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance[END_REF]. Interestingly, enzymes such as the cortex lytic enzyme SleC are sensitive to the presence of δ-lactams (see C. difficile spore germination section) [START_REF] Francis | Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination[END_REF][START_REF] Francis | Detecting Cortex Fragments During Bacterial Spore Germination[END_REF][START_REF] Gutelius | Functional analysis of SleC from Clostridium difficile: an essential lytic transglycosylase involved in spore germination[END_REF]. Another distinctive characteristic of the C. difficile cortex is that 22% of the muropeptides in the cortex were not substituted, in contrast with B. subtilis where most of them were substituted with L-alanine. Finally, 55% to 61% of the muropeptides were N-deacetylated in the cortex of C. difficile 630erm whereas no or very low N-deacetylation was observed in the cortex of B. subtilis or C. perfringens (10%) [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF][START_REF] Popham | Analysis of the peptidoglycan structure of Bacillus subtilis endospores[END_REF][START_REF] Orsburn | Factors contributing to heat resistance of Clostridium perfringens endospores[END_REF]. This high percentage of N-deacetylation in the cortex of C. difficile may be driven by the high number of N-deacetylases. One of the enzymes potentially involved in the spore cortex N-deacetylation is PgdB (CD630_32570) [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF]. Indeed, pgdB was poorly expressed during the bacterial growth and is under control of  E factor [START_REF] ** Coullon | N-Deacetylases required for muramic-δ-lactam production are involved in Clostridium difficile sporulation, germination, and heat resistance[END_REF][START_REF] Saujet | Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile[END_REF]. Moreover, a slight decrease in glucosamine N-deacetylation (5%) of the spore cortex was observed in the ΔpgdB mutant [START_REF] ** Coullon | Peptidoglycan analysis reveals that synergistic deacetylase activity in vegetative Clostridium difficile impacts the host response[END_REF]. This suggest that other N-deacetylases are involved in the N-deacetylation of the spore cortex in C. difficile. Spore cortex is synthesized by both the mother cell and the forespore [START_REF] Popham | Spore Peptidoglycan[END_REF][START_REF] Vasudevan | Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K[END_REF]. During the engulfment stage of sporulation, the bacterial peptidoglycan is separated from the spore cortex by specific autolysins such as SpoIID and SpoIIP [START_REF] Diaz | Clostridium difficile Lipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination. mSphere 2018, 3. This publication analyzed the synthesis of δ-lactams in the spore cortex of C. difficile by GerS, CwlD and PdaA 38[END_REF][START_REF] Dembek | Peptidoglycan degradation machinery in Clostridium difficile forespore engulfment[END_REF][START_REF] Kelly | (B) structures of C. difficile are presented. A minimal structure named muropeptide is represented[END_REF] (see C. difficile spore assembly section), and this process relies on the structural differences between bacterial peptidoglycan and spore cortex. Since very little is known about N-deacetylation in the spore cortex, the impact of this modification has to be further investigated to fully understand its contribution to the sporulation and germination processes. Conclusions Some advances have been highlighted in studying the peptidoglycan and cortex of C. difficile in the last decade. A deeper study of the peptidoglycan structure, biogenesis, recycling and degradation will allow a better understanding of the role of the C. difficile peptidoglycan in the toxin release and in the antibiotic and antimicrobial resistances. Finally, the recent analysis of the cortex structure will allow a better understanding of the germination process through its hydrolysis.

FundingHFigure 1 :

 1 Figure 1: Peptidoglycan and cortex structures Peptidoglycan (A) and cortex (B) structures of C. difficile are presented. A minimal structure named muropeptide is represented in the right-hand side framed section of the figure. It is composed of two sugars, a muramic acid and a glucosamine, and a peptide which contains L-alanine (L-Ala), D-glutamate (D-Glu), 2,6 diaminopimelic acid (A2pm) and D-Alanine (D-Ala) as represented. Peptidoglycan and cortex structures are represented here by assembling of typical muropeptides linked together, forming a chain of glycan. The principal modifications are indicated and highlighted by a colored box.