Transito Elodie Cuenot

Thibaut Douche

Olivier Gorgette

Pascal Courtin

Sandrine Denis-Quanquin

Sandra Hoys

Yannick D N Tremblay

Mariette Matondo

Marie-Pierre Chapot-Chartier

Transito Garcia-Garcia

Claire Janoir

Bruno Dupuy

Thomas Candela

Isabelle Martin-Verstraete

The Ser/Thr kinase PrkC participates in cell wall homeostasis and antimicrobial resistance in Clostridium difficile

Keywords: Hanks kinase, cephalosporin sensitivity, cell morphology, envelope homeostasis, CAMPs, biofilms, peptidoglycan

Introduction

Clostridium difficile is the leading cause of antibiotic-associated infections in adults.

Severity of C. difficile infection (CDI) symptoms range from diarrhea to life-threatening pseudo-membranous colitis. Major risks associated with CDI include antibiotic exposure leading to dysbiosis of gut microbiota, as well as, advanced aged and hospitalization (1).

The impact of CDI is significant in terms of mortality, morbidity, disease management and financial burden. With the emergence of new isolates, the incidence and severity of CDI have increased both in North America and Europe [START_REF] Smits | Clostridium difficile infection[END_REF]. C. difficile is acquired from the environment through the ingestion of spores. This resistant form of the bacterium is thought to be responsible for transmission, environmental persistence and dissemination. When the normal intestinal microbiota is disrupted, the pools of metabolites present in the gut change leading to an increased concentration of cholate conjugates, which in turn triggers spore germination [START_REF] Abt | Clostridium difficile colitis: pathogenesis and host defence[END_REF][START_REF] Sorg | Microbial bile acid metabolic clusters: the bouncers at the bar[END_REF]. The vegetative cells can then multiply and colonize the dysbiotic gastrointestinal tract. Toxigenic strains of C. difficile produce two toxins, TcdA and TcdB, which are its main virulence factors. These toxins cause alteration of the actin cytoskeleton of epithelial cells and neutrophil recruitment causing local inflammation [START_REF] Abt | Clostridium difficile colitis: pathogenesis and host defence[END_REF]. Following the administration of a targeted antibiotic therapy such as metronidazole or vancomycin, CDI relapse can occur, involving spore and possibly biofilm formation [START_REF] Pantaleon | Biofilms of Clostridium species[END_REF][START_REF] Paredes-Sabja | Clostridium difficile spore biology: sporulation, germination, and spore structural proteins[END_REF]. Additional factors including those involved in stress adaptation and surface associated proteins, such as adhesins, the fibronectin-binding protein (FbpA), the surface layer protein (SlpA) and the flagellum also participate in the colonization process, allowing C. difficile to establish its intestinal niche [START_REF] Janoir | Virulence factors of Clostridium difficile and their role during infection[END_REF].

Gut dysbiosis is an essential step that allows C. difficile to colonize the colon and cause an infection. Certain antibiotics such as cephalosporins, clindamycin and fluoroquinolones are known to increase the risk of developing a CDI. Most of C. difficile strains are resistant to these antibiotics, which is of major concern (1). Fluoroquinolone resistance in C. difficile strains is associated with amino acid substitution in the gyrases GyrA or GyrB (1). The mechanisms responsible for cephalosporin resistance by C. difficile remain poorly characterized (1). However, some progress has been made, as a class D ß-lactamase was identified in the genomes of most C. difficile strains [START_REF] Toth | Intrinsic Class D beta-Lactamases of Clostridium difficile[END_REF]. In addition to antibiotics, vegetative cells also encounter secondary bile salts (4), host secreted antimicrobial peptides and also reactive oxygen and nitrogen species produced during inflammation [START_REF] Abt | Clostridium difficile colitis: pathogenesis and host defence[END_REF][START_REF] Kint | The alternative sigma factor sigma(B) plays a crucial role in adaptive strategies of Clostridium difficile during gut infection[END_REF]. To survive in the host, C. difficile must detect the host environment and induce an appropriate survival strategy, which may include activating the general stress response, sporulation, biofilm formation, and pro-ducing virulence factors [START_REF] Pantaleon | Biofilms of Clostridium species[END_REF][START_REF] Paredes-Sabja | Clostridium difficile spore biology: sporulation, germination, and spore structural proteins[END_REF][START_REF] Kint | The alternative sigma factor sigma(B) plays a crucial role in adaptive strategies of Clostridium difficile during gut infection[END_REF]. Protein phosphorylation is a reversible post-translational modification used to transduce signal and regulate cellular processes. Bacterial serine/threonine kinases of the Hanks family (STKs) share structural similarities with eukaryotic serine/threonine kinases and undergo substrate phosphorylation on serine or threonine residues [START_REF] Pereira | Eukaryote-like serine/threonine kinases and phosphatases in bacteria[END_REF]. STKs and their associated phosphatases (STP) regulate numerous physiological processes including translation, carbon and cell-wall metabolism, antibiotic tolerance, cell division, developmental processes, and virulence [START_REF] Pereira | Eukaryote-like serine/threonine kinases and phosphatases in bacteria[END_REF][START_REF] Dworkin | Ser/Thr phosphorylation as a regulatory mechanism in bacteria[END_REF]. Phosphoproteomic studies revealed that STKs might phosphorylate a broad spectrum of substrates [START_REF] Jers | Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks[END_REF] including enzymes, components of the cellular machinery involved in translation, division or repair and also several transcriptional regulators that mainly form part of two-component systems [START_REF] Pereira | Eukaryote-like serine/threonine kinases and phosphatases in bacteria[END_REF][START_REF] Pompeo | Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis[END_REF]. Therefore, STKs are key integration nodes in signaling networks that control bacterial physiology, growth and stationary phase.

In B. subtilis, PrkC is a trans-membrane kinase with an extracellular signal receptor domain containing penicillin-binding and STK associated (PASTA) repeats. PrkC interacts with peptidoglycan fragments and ß-lactam in vitro [START_REF] Maestro | Recognition of peptidoglycan and beta-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae[END_REF][START_REF] Squeglia | Chemical basis of peptidoglycan discrimination by PrkC, a key kinase involved in bacterial resuscitation from dormancy[END_REF], while a PASTA-STK (Stk1) homologue in Staphylococcus aureus interacts with lipid II [START_REF] Hardt | The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus[END_REF]. In B. subtilis, PrkC is a key-signaling enzyme that controls stationary phase survival, biofilm formation, sporulation, germination [START_REF] Pompeo | Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis[END_REF] and peptidoglycan (PG) metabolism [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF]. Inactivation of genes encoding PASTA domain-containing STKs are associated with a range of phenotypes among Gram-positive bacteria, with defects in cell division and changes in cell-wall metabolism commonly observed [START_REF] Pereira | Eukaryote-like serine/threonine kinases and phosphatases in bacteria[END_REF][START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF][START_REF] Pensinger | Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets[END_REF]. In S. aureus, Stk1 plays a role in virulence, antibiotic resistance and cell-wall remodeling [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF] whereas in Streptococcus pneumonia, StkP controls cell division, competence, virulence, adherence to eukaryotic cells, stress response and cell wall metabolism [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF][START_REF] Banu | The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism[END_REF].

The role of STKs has not been studied in anaerobic firmicutes. C. difficile has two genes encoding STKs, CD2148 and prkC (CD2578) and one gene encoding a STP (CD2579). In this study, we characterized the role of the C. difficile PASTA-STK, PrkC, in the regulation of the physiological processes important in CDI. Inactivation of the prkC gene resulted in changes in the morphology and the properties of the cell envelope as well as increased sensitivity to antimicrobial compounds targeting the cell wall.

Materials and methods

Bacterial strains and growth conditions

C. difficile strains and plasmids used in this study are listed in Table S1. C. difficile strains were grown anaerobically (5% H2, 5% CO2, 90% N2) in TY broth (Bacto tryptone 30 g.l -1 , yeast extract 20 g.l -1 , pH 7.4), in Brain Heart Infusion broth (BHI) or in a peptone-containing medium (Pep-M) [START_REF] Ng | Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles[END_REF]. Sporulation medium (SM) [START_REF] Wilson | Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile[END_REF] was used for sporulation assays and spores were produced in SMC medium [START_REF] Dembek | Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores[END_REF]. Solid media were obtained by adding agar to a final concentration of 17 g.l -1 . When necessary, cefoxitin (Cfx, 25 μg.ml -1 ) or thiamphenicol (Tm, 15 μg.ml -1 ) was added. E. coli strains were grown in LB broth. When indicated, ampicillin (100 μg.ml -1 ) or chloramphenicol (15 μg.ml -1 ) was added. Anhydrotetracycline (ATc) was used to induce expression of the prkC gene from the Ptet promoter of pDIA6103 [START_REF] Fagan | Clostridium difficile has two parallel and essential Sec secretion systems[END_REF].

Construction of plasmids and strains

The allelic exchange cassette used to delete of the CD2578 gene (prkC) was constructed in a derivative of pMTLSC7315 ( 24) lacking the multi-cloning site. DNA sequences flanking the prkC gene (1038 and 1117-bp) were amplified by PCR using primers IMV854 and IMV855 or IMV856 and IMV857 (Table S2 andFig To construct a translational SNAP-PrkC fusion, we amplified the SNAP coding sequence fused to a linker in 3' (GGATCCGCAGCTGCT) using pFT58 as a template and primers EC238 and EC239. pDIA6413 (pDIA6103-prkC) was amplified by inverse PCR using the primers EC236 and EC237. Using these two fragments, pDIA6855 was obtained by Gibson Assembly. pDIA6855 was conjugated in the 630∆erm strain to obtain CDIP1357 (Table S1).

To determine if prkC is co-transcribed with upstream genes (CD2579 and rlmN), we carried out an RT-PCR experiment using RNA extracted from exponentially growing cells. After cDNA synthesis using IMV843, a primer targeting the prkC gene, PCR amplification was tested with pairs of primers located in adjacent genes (prkC/CD2579 or CD2579/rlmN).

Non-treated RNAs were used as a negative control.

Antimicrobial sensitivity tests

Cultures of C. difficile strains (OD600nm of 0.3) were plated on BHI agar plates. 6-mm paper disks containing antibiotics were placed onto the agar surface. Lysozyme (800 μg) was also placed on a 6-mm paper disk using Pep-M agar plates. The growth inhibition diameter was measured after 24 h of incubation at 37°C. Minimal Inhibitory Concentrations (MICs) were determined on BHI plates by E-test (bioMérieux) after 24 h incubation at 37°C. To test susceptibility for antimicrobial peptides, protocols were modified from previous published work [START_REF] Ho | Clostridium difficile extracytoplasmic function sigma factor sigmaV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF][START_REF] Mcbride | The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile[END_REF]. Each well of a 24-well microplate was inoculated with 1 ml of a BHI bacterial inoculum (OD600nm of 0.01). After addition of bacitracin (Sigma Aldrich), polymixin B (Sigma Aldrich) or nisin (Sigma Aldrich), microplates were incubated at 37°C for 20 h. The MIC was defined as the lowest concentration of antimicrobial peptide preventing growth. 5 μl of an overnight culture of C. difficile strains was streaked on BHI plates without or with SDS (0.005% to 0.009%). The plates were incubated at 37°C for 48 h. Deoxycholate (DOC) resistance tests were performed in 24-well microplates. Each well was inoculated with 1 ml of TY with or without DOC (0.03%). The OD600nm of each culture was monitored. For autolysis, cells (OD600nm of 1) were resuspended in 50 mM potassium phosphate buffer (pH 7.0) containing 0.01 % of Triton X-100 and incubated at 37°C. The OD600nm was determined every 5 min.

Sporulation, germination, motility and biofilm formation assays

Sporulation assays were performed as described previously [START_REF] Saujet | The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile[END_REF]. To purify spores, 100 μl of culture plated on solid SMC were grown at 37°C for 7 days. Spores were scraped off with water and then incubated 7 days at 4°C to allow the release of spores from the cells. Spores were purified by centrifugation using a HistoDenz (Sigma-Aldrich) gradient as described previously [START_REF] Dembek | Transcriptional analysis of temporal gene expression in germinating Clostridium difficile 630 endospores[END_REF]. For the germination efficiency tests, we monitored OD600nm of purified spores incubated under anaerobiosis in BHIS supplemented with 0.5 % taurocholate, with bryostatin or muropeptides.

To measure motility, plates containing 25 ml of BHI agar (0.3% w/v), Tm and ATc were inoculated with 5 μl of an exponential-phase culture. Plates were incubated for 48h at room temperature and the zone of motility was then measured. For the biofilm assay, 1 ml of BHI-S medium containing 0.1 M glucose, 0.1% cysteine and polymixin B (20 μg.ml -1 ) or DOC (0.01%) was inoculated and deposited in a well of a 24-well microplate. Microplates were incubated at 37°C. The biofilm was washed with PBS (Phosphate buffered saline), stained with 1 ml of crystal violet (0.2%) followed by two washes with PBS. The OD570nm was measured after resuspension of the cells in methanol/acetone (80%/20%) using noninoculated medium as a negative control. For dispersion of preformed biofilms, 24 h and 48 h biofilms were treated with DNase I (100 μg/ml in 150 mM NaCl, 1 mM CaCl2) under anaerobic conditions at 37°C for 1 h [START_REF] Dubois | A microbiota-generated bile salt induces biofilm formation in Clostridium difficile[END_REF]. Control wells were treated with buffer without DNase. Biofilms were then washed, stained, and quantified as described above.

Phase contrast and transmission electron microscopy

C. difficile strains were grown 5 h in TY medium. For phase contrast microscopy, cells were analyzed using an axioskop microscope (Zeiss). For transmission electron microscopy (TEM), cultures were mixed with 1 volume of a fixative solution containing 4% paraformaldehyde (PFA) and 4% glutaraldehyde (GA) in PHEM buffer 1x pH 7.3 (60 mM Pipes, 25mM Hepes, 10 mM EGTA, 2mM MgCl2) and then incubated for 30 min at room temperature. After centrifugation, pellets were resuspended in a second fixative solution (2% PFA and 2% GA in PHEM buffer pH= 7.3) and incubated for 1 h. Samples were then washed twice in PBS prior to being subjected to a high pressure (>2000 bar) freezing in 1hexadecane using a BAL-TEC HPM 010 (LEICA). Freeze substitution was done with 2% OsO4 in acetone followed by several steps: -90°C for 42 h, warmed up to -30°C (5°C/h), incubated for 12 h, warmed up to 0°C (10°C/h) and incubated for 1h. Samples were then washed with acetone on ice and incubated in EPON/acetone at different volume ratio (1/3 for 3 h, 1/1 for 3 h; 2/1 ON; 3/1 for 4 h), followed by an incubation in pure EPON (2 h, overnight; 6 h). Samples were then incubated overnight in EPON and the hardener BDMI prior to polymerization at 60°C for 48 h. Sections (60-70 nM) were obtained on a FC6/UC6 ultramicrotome (Leica), transferred on 200 Mesh Square Copper grids coated with formvar and carbon (CF-200-Cu50, Delta Microscopy). Samples were stained with 4% uranyl acetate and counterstained with lead citrate. Images were recorded with TECNAI SPIRIT 120 kv (with a bottom-mounted EAGLE 4Kx4K Camera).

SNAP labelling, fluorescence microscopy and image analysis

For membrane and chromosome staining, 500 μl of exponential-phase cells were centrifuged and resuspended in 100 μl of PBS containing the fluorescent membrane dye FM 4-64 (1 μg/ml, Molecular Probes, Invitrogen) and the DNA stain 4,6-Diamidino-2-phenylindole (DAPI) (2 μg/ml, Sigma). Samples were incubated for 2 min in the dark and mounted on 1.2% agarose pad. Strain CDIP1357 was grown 2 h in TY and expression of the SNAP Cd -prkC fusion was induced with 50 ng/ml of ATc for 2 h. For SNAP labelling, the TMR-Star substrate (New England Biolabs) was added at 250 nM, and the mixture was incubated for 30 min in the dark under anaerobiosis. Cells were then collected by centrifugation, washed and resuspended in PBS. Cell suspension (3 μl) was mounted on 1.7% agarose-coated glass slides. The images were taken with 300 ms exposure times for the autofluorescence and 900 ms exposure times for the SNAP using a Nikon Eclipse TI-E microscope 100x Objective and captured with a CoolSNAP HQ2 Camera. The images were analysed using ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF].

Detection of phospho-threonine by western blot on soluble and insoluble fractions.

Cells were grown for 6 h in TY and then harvested by centrifugation. The pellets were resuspended in PBS containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich) and 0.12 μg/ml of DNase. Cells were lysed for 45 min at 37°C and then centrifuged. The soluble fraction was diluted twice with 2X Sample buffer (150 mM Tris-HCl pH6.8, 30% glycerol, 1.5 % SDS, 15% β-mercaptoethanol, 2 μg/ml of bromophenol blue). After 2 washes with PBS, the insoluble fraction was resuspended in 2X PBS containing 1% SDS, and mixed with 2X Sample buffer. Western blots were performed using an anti-P-Thr primary antibody (Cell signaling) followed by a goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody (Sigma-Aldrich) and developed using the SuperSignalWest Femto chemiluminescent Kit (Thermo scientific).

Peptidoglycan and polysaccharide II analysis

Peptidoglycan (PG) samples were prepared from 1.5 l of C. difficile grown in BHI (OD600nm of 1) as previously described [START_REF] Peltier | Clostridium difficile has an original peptidoglycan structure with a high level of Nacetylglucosamine deacetylation and mainly 3-3 cross-links[END_REF]. Purified PG was then digested with mutanolysin (Sigma Aldrich) and the soluble muropeptides were separated by reverse phase high-performance liquid chromatography (RP-HPLC) [START_REF] Peltier | Clostridium difficile has an original peptidoglycan structure with a high level of Nacetylglucosamine deacetylation and mainly 3-3 cross-links[END_REF]. Polysaccharides II (PSII) were extracted from C. difficile grown in BHI (OD600nm of 1) as previously described [START_REF] Reid | Structural characterization of surface glycans from Clostridium difficile[END_REF]. PSII was lyophilized and its structure was checked by [ 1 H] and [ 13 C] NMR. Spectra were acquired on a 400 MHz Bruker spectrometer equipped with a Prodigy probe. PSII was lyophilized and its structure was determined by [ 1 H] NMR. For the quantification of PG and PSII, 500 ml of culture (OD600nm of 1) was used to purify PSII covalently linked to PG (PG-PSII). PG-PSII was purified as previously described [START_REF] Candela | Bacillus anthracis CapD, belonging to the gammaglutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan[END_REF] without the acetone treatment. Linkage between PG and PSII was disrupted after an incubation for 48 h at 4°C in hydrofluoric acid (48%). The pellet containing PG was washed three times in H2O and the hydrofluoric acid supernatant that contains PSII subunits was evaporated and resuspended in H2O. Both, PG and PSII were lyophilized and then weighted.

For the PSII dot blot, exponential phase cultures were harvested by centrifugation. Supernatant and total crude cell extracts were kept as separate fractions. The supernatant fraction was recovered and precipitated with 10% TCA for 30 min. The supernatant and the total crude cell fractions were treated with 100 g/ml of proteinase K (Sigma) for 1 h at 37°C. Samples were then serially diluted and 5 μl of each dilution were spotted onto an activated polyvinylidene difluoride membrane. The membrane was washed in H2O, blocked for 15 min in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20, pH7.5) containing 10% milk, and then washed in 5% milk in TBST for 2 min. After overnight incubation in PSII-LTB rabbit antiserum (1:8,000) (34), the membrane was washed once in TBST with 5% milk, twice in TBST for 5 min, and once in TBST with 5% milk for 10 min. Following incubation with goat anti-rabbit horseradish-peroxidase-conjugated secondary antibody at 1:10,000 dilution for 1 h, the membrane was washed 5 times in TBST for 5 min, and revealed using the SuperSignalWest Femto chemiluminescent substrate.

Isolation of cell-wall proteins and proteomic analysis

C. difficile strains were grown for 6 h in TY at 37°C and 20 mL of each culture was then centrifuged. The cell pellets were washed with PBS, resuspended (OD600nm of 100) in 75 mM Tris-HCl pH6.8, 15% glycerol, 7.5% β-mercaptoethanol, 0.75% SDS and boiled for 10 min at 100°C. Proteins were precipitated with 10% TCA, washed with 90% cold acetone, dried, resuspended in urea 8 M/NH4HCO3 100 mM and sonicated. Total protein extracts (50 µg) were reduced with TCEP (10 mM) (Sigma) for 30 min and alkylated with iodoacetamide 20 mM (Sigma) for 1 h. Proteins were digested with rLys-C 1 µg (Promega) for 4 h at 37°C and with Trypsin 1µg (Promega) overnight at 37°C. The digestion was stopped with Formic acid (FA) 4%. Peptides were desalted on C18 Sep-Pak Cartridge (WAT054955, Waters) and eluted with Acetonitrile (ACN) 50%/FA 0.1% and then ACN 80%/FA 0.1% before being dried in vacuum centrifuge. Peptides were resuspended with ACN 2%/FA 0.1%. A nanochromatographic system (Proxeon EASY-nLC 1000, Thermo Fisher Scientific) was coupled on-line to a Q Exactive TM HF Mass Spectrometer (Thermo Fisher Scientific). Peptides (1 μg) were injected onto a 47 cm C18 column (1.9 μm particles, 100 Å pore size, ReproSil-Pur Basic C18, Dr. Maisch GmbH) and separated with a gradient from 2 to 45 % ACN at a flow rate of 250 nl/min over 132 min. Column temperature was set to 60°C. Data were ac-quired as previously described [START_REF] Lago | Proteome remodelling by the stress sigma factor RpoS/sigma(S) in Salmonella: identification of small proteins and evidence for post-transcriptional regulation[END_REF]. Raw data were analyzed using MaxQuant software version 1.5.1.2 (36) using the Andromeda search engine [START_REF] Cox | Andromeda: a peptide search engine integrated into the MaxQuant environment[END_REF]. The MS/MS spectra were searched as previously described ( 35) against an internal C. difficile database containing 3,957 proteins and the contaminant file included in MaxQuant. The statistical analysis was performed with Perseus 1.5.2.6 (38) as previously described [START_REF] Lago | Proteome remodelling by the stress sigma factor RpoS/sigma(S) in Salmonella: identification of small proteins and evidence for post-transcriptional regulation[END_REF]. Missing values for LFQ intensities were imputed and replaced by random LFQ intensities that are drawn from a normal distribution at the low detection level. Statistical significance was assessed with a two-sided t-test of the Log2 transformed LFQ intensities with a permutation-based FDR calculation at 5% and S0=2 [START_REF] Tusher | Significance analysis of microarrays applied to the ionizing radiation response[END_REF]. Differentially regulated proteins are visualized on a Volcano-Plot. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (40) partner repository with the dataset identifier PXDX012241.

Golden Syrian hamster infections

Golden Syrian hamsters were first treated with a single oral dose of 50 mg/kg of clindamycin. Five days after the antibiotics treatment, the hamsters were infected by gavage with 5000 spores of either the 630∆erm strain or the ∆prkC mutant. Spore inocula were standardized before challenge. Eight animals per strain per experiment were used for the infection and we performed two independent experiments. Colonization was followed by enumeration of C. difficile cells in feces samples and was started 2-day post infection and each day until the death of the animal. Briefly, feces were resuspended in PBS at 10 mg.ml -1 and serially diluted with PBS before plating on BHI supplemented with 3% of defibrinated horse blood and the C. difficile selective supplement containing cycloserine (25 µg/ml) plus cefoxitine (8 µg/ml). All animal experiments were conducted according to the European Union guidelines for the handling of laboratory animals and Procedures for infection, euthanasia, and specimen collection were approved by the Central Animal Care Facilities and Use committee of University Paris-Sud (agreement 92-019-01; protocol number 2012-107).

Results

CD2578 is the

The prkC locus of C. difficile

The prkC gene likely belongs to a large cluster of genes, which ranges from dapF (CD2590) to CD2578-prkC (Fig 2A). As usually observed in other firmicutes [START_REF] Iwanicki | Transcription in the prpC-yloQ region in Bacillus subtilis[END_REF][START_REF] Kristich | A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence[END_REF], a gene (CD2579-stp) encoding a PP2C-type phosphatase (STP), which is probably involved in the dephosphorylation of the PrkC substrates, is adjacent to prkC. Upstream of stp, we found genes encoding proteins involved in translation (rlmN, rsmB, def and fmt), transcription (rpoZ), DNA replication (priA) and metabolism (gmk and coaBC). This cluster is conserved in B. subtilis and B. cereus with the exception of 3 genes that are only present in C. difficile; CD2582 and CD2583 encoding membrane proteins and dapF encoding a diaminopimalate epimerase (Fig 2A). To determine if prkC is co-transcribed with stp as observed in other firmicutes, and the other genes located upstream of stp such as the rlmN gene, RT-PCR experiments were performed. PCR products were detected using primer pairs located in adjacent genes (prkC/stp or stp/rlmN) with RNAs processed by a reverse transcriptase but not with untreated RNA used as negative control (Fig 2B). This result indicated that the prkC gene is in operon with stp but also rlmN. Using a genome-wide transcriptional start site (TSS) mapping [START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF], we identified a unique TSS in the prkC locus located 23 bp upstream of dapF suggesting that this gene might be the first gene of a large operon. We found a TG-N-TATAAT extended -10 box specific for the consensus of σ A -dependent promoters upstream of the TSS (Fig 2A). However, we cannot exclude the existence of additional promoters between dapF and prkC that were not identified in the genome-wide TSS mapping [START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF].

Localization of the PrkC protein

To determine the cellular location of PrkC, we expressed the prkC gene fused to a HA tag under the control of the inducible promoter Ptet (pDIA6103-Ptet-prkC-HA). A western blot analysis of the membrane or cytoplasmic fraction with an anti-HA antibody revealed two bands corresponding to proteins of about 100 kDa and 50 kDa (Fig S2A). These two proteins were only detected in the membrane fraction (Fig 1B). The expected molecular weight of the PrkC-HA tagged protein is around 75 kDa and the band detected around 100 kDa likely corresponds to PrkC-HA. Indeed, the PrkC and StkP proteins of B. subtilis and S. pneumoniae are detected with an apparent molecular weight higher than their expected size [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF][START_REF] Morlot | Interaction of Penicillin-Binding Protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae R6 morphogenesis[END_REF] and the phosphorylation of the kinase results in a reduced electrophoretic mobility [START_REF] Kristich | Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis[END_REF]. The lower band at 50 kDa could be a form of degradation product that might correspond to the extracellular domain fused to HA. It is worth noting that PrkC of B. subtilis is sensitive to protease cleavage [START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF].

To determine the localization of PrkC, we used a 630∆erm strain containing a plasmid encoding a SNAP Cd -PrkC protein fusion produced under the control of the Ptet promoter. The production of the SNAP Cd -PrkC protein is stable as shown by western blot using an anti-SNAP antibody (Fig S2B). After 2 h of induction, we detected the SNAP Cd -PrkC fusion protein fusion at the septum of dividing cells (Fig 1C). Thus, the PrkC protein is membrane associated and localizes at the septum during cell growth.

Deletion of the prkC gene in C. difficile and detection of PrkC activity

To study the role of PrkC in the physiology of C. difficile, we inactivated the prkC gene by allelic exchange in the strain 630∆erm [START_REF] Cartman | Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production[END_REF]. Deletion of the prkC gene from codon two to the stop codon was confirmed by PCR (Fig S1). The prkC mutant was complemented with the wild-type prkC gene (pDIA6103-Ptet-prkC) or a modified copy of prkC (pDIA6103-Ptet-prkC-K39→A) with the lysine residue required for phosphotransfer replaced by an alanine.

The kinase activity of PrkC was demonstrated by comparing the profiles of phosphorylation of the wild-type (WT) strain or the ∆prkC mutant carrying either pDIA6103, pDIA6103-Ptet-prkC or pDIA6103-Ptet-prkC-K39→A. We assumed that PrkC and PrkC-K39→A were produced to the same level and were located in membrane because we detected similar levels in the membrane fraction of these proteins with an HA tag fusion (PrkC-HA or PrkC-K39→A-HA) (Fig S2A). We detected proteins phosphorylated on threonine in the WT strain both in soluble and insoluble fractions by western blot analysis with an antibody against phosphorylated threonine residues (α-PThr (Fig 1D). Some bands that were detected in the WT strain and with a lower intensity in the complemented strain disappeared in the ∆prkC mutant and in the strain producing a modified PrkC-K39→A protein. This result strongly suggested that PrkC has a kinase activity in vivo and that the lysine at position 39 (Fig 1A) is essential for the phosphorylation of PrkC targets as observed in the PASTA-STKs of other firmicutes [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF][START_REF] Huse | The conformational plasticity of protein kinases[END_REF].

Impact of prkC deletion on growth, sporulation and germination

The 630∆erm strain and the ∆prkC mutant exhibited nearly identical doubling time and growth yield in TY (Fig S3A). No difference in CFU was also observed between the two strains (Fig S4A and data not shown). We also performed co-culture of the WT and ∆prkC mutant strains in TY (Fig S3C ). We observed that the ∆prkC mutant was less abundant than the wild-type strain by two-fold after 8 h or 24 h of growth. This suggests that there is no drastic difference in fitness associated with the inactivation of prkC. The growth of the ∆prkC containing pDIA6103-Ptet-prkC was similar to that of the WT strain in the presence of 15 ng/ml of ATc but was affected when we added 50 ng/ml of ATc (Fig S3B). This growth defect was also observed in the ∆prkC mutant expressing prkC-K39→A (Fig S3B). In contrast, the addition of increasing concentrations of ATc had no effect on the growth of the WT strain harboring an empty plasmid (Fig S3B ). A qRT-PCR analysis also showed that expression of prkC was similar in the WT and complemented strains in the presence of 15 ng/ml of ATc (Fig S3D). In the presence of 50 ng/ml of ATc, the expression of prkC probably increased in the ∆prkC strain containing pDIA6103-Ptet-prkC because we detected a drastic overproduction of the SNAP-PrkC fusion in the presence of 50 ng/ml when compared to 20 ng/ml (Fig S2). Over-production of PrkC is likely responsible for the growth defect observed in the presence of 50 ng/ml of ATc suggesting that a proper level of prkC expression is important for the fitness of C. difficile. Since the kinase activity of PrkC is apparently not responsible for the growth defect when the protein is overexpressed, the trans-membrane segment or the extracellular domain are probably involved in this toxicity.

To determine if PrkC is involved in sporulation, we measure sporulation levels of the WT and the ∆prkC mutant strains after 24 h or 72 h of growth in SM. No significant differences were observed between both strains for the quantity of spores produced (Fig S4A). When we tested the ability of purified spores to germinate, we observed that the drop in the OD600nm of spore suspensions after the addition of taurocholate was similar for the WT and the ∆prkC mutant (Fig S4 B). In B. subtilis, PrkC is involved in the germination of spores in the presence of B. subtilis muropeptides [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. However, purified muropeptides from C. difficile (0.1 to 0.01 mg/ml) failed to induce spore germination of the WT or the ∆prkC mutant (data not shown). Bryostatin is a molecule involved in STK activation that induces germination of B. subtilis spores [START_REF] Shah | A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments[END_REF]. This compound at 1 µM or 10 µM had no effect on C. difficile spores (data not shown). All these results indicated that PrkC is not involved in the control of sporulation and spore germination in C. difficile.

prkC deletion affects cell morphology

To determine if deletion of prkC had an effect on the cell morphology, we analyzed by phase contrast microscopy cells of the 630∆erm strain, the ∆prkC mutant, and the complemented strain during exponential growth phase (Fig S5A). The ∆prkC mutant cells were more elongated than those of the WT strain and the complemented strain (Fig S5A). We then measured the cell size using cells labeled with DAPI and FM4-64 (Fig 3A and3B). The ∆prkC mutant cells had an average cell size of 9.2 μm whereas the WT and the complemented strains had an average cell size of 4.5 μm and 6.

prkC deletion increases sensitivity to detergents and autolysis

To determine if prkC deletion also affects the cell envelop, we tested the sensitivity of the prkC mutant to compounds with a detergent activity. The presence of 0.006% SDS affected the growth of the ∆prkC mutant but did not affect the WT and the complemented strains (Fig 5A). It is worth noting that in the gastrointestinal tract, C. difficile is subjected to the bactericidal effects of secondary bile salts that solubilize phospholipids, impair membrane integrity and weaken cell wall [START_REF] Hofmann | Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics[END_REF]. We therefore tested the sensitivity of the ∆prkC mutant to the secondary bile salt, deoxycholate (DOC) and to the primary bile salt, cholate. While the sensitivity to cholate (0.03% or 0.04 % in TY) was similar for all the strains (data not shown), we observed that the growth of the ∆prkC mutant was reduced in the presence of 0.03% DOC (Fig 5B ), a physiological concentration encountered in the gut [START_REF] Northfield | Postprandial concentrations of free and conjugated bile acids down the length of the normal human small intestine[END_REF][START_REF] Ridlon | Bile salt biotransformations by human intestinal bacteria[END_REF]. In addition, the autolysis in the presence of 0.01% of the non-ionic detergent Triton X-100 is more rapid for the ∆prkC mutant than for the WT and the complemented strains (Fig 5C ) while no difference in autolysis was observed in the absence of Triton (Fig S6A). This result suggested that the ∆prkC mutant is more sensitive to the PG hydrolysis.

PrkC controls sensitivity to antimicrobial compounds targeting the cell envelop

Given that the deletion of prkC affected cell morphogenesis, we tested the sensitivity of the prkC mutant to antibiotics targeting cell wall biosynthesis [START_REF] Kristich | Reciprocal regulation of cephalosporin resistance in Enterococcus faecalis[END_REF][START_REF] Beltramini | Modulation of cell wall structure and antimicrobial susceptibility by a Staphylococcus aureus eukaryote-like serine/threonine kinase and phosphatase[END_REF]. Using disk assays, we showed that the size of the zone of inhibition increased for the ∆prkC mutant compared to the WT and the complemented strains for all ß-lactams tested (Fig 6A). Thus, the prkC mutant is more susceptible to ticarcillin, amoxicillin, imipenem and several cephalosporins (cefoxitin, ceftazidime and cefepime). In contrast, we did not observe any differences for antibiotics that target transcription or translation (Fig 6A and Fig S6B). We also observed an important reduction in the MIC of the ∆prkC mutant compared to the WT strain for the glycopeptide, teicoplanin (>12-fold), but not for vancomycin (2-fold) (Table 1). In addition, we observed a slight increase in the susceptibility of the mutant to amoxicillin and imipenem (4fold) and a more substantial increase for the cephalosporins tested (Table 1). The MIC for the second-generation cephalosporin, cefoxitin, was reduced by more than 6-fold for the ∆prkC mutant compared to the WT strain whereas the MIC for the third generation cephalosporins, ceftazidime, cefepime and cefotaxime, was reduced for the ∆prkC mutant by 20-17.5-and 8-fold, respectively. The same increase in sensitivity to cefoxitin, ceftazidine and cefepime was seen for the ∆prkC mutant carrying pDIA6103-Ptet-prkC-K39→A (Fig 6A ), suggesting that the kinase activity of PrkC is required for C. difficile to express an intrinsic high-level of resistance to cephalosporins (1). Cationic antimicrobial peptides (CAMPs) that are produced by both bacteria and the host also target the cell envelop and/or membrane of bacteria [START_REF] Nawrocki | Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria[END_REF]. Thus, we tested the resistance of the WT and mutant strains for several CAMPS of bacterial origin. The MIC of the ∆prkC mutant for polymixin B, bacitracin and nisin was 6-, 22-and 4.5-fold lower, respectively than the MIC for the WT strain (Table 1). In the gastrointestinal tract, host-produced antimicrobial proteins such as lysozyme inhibit colonization of many Gram-positive pathogens by hydrolyzing the PG and breaking the integrity of the cell wall. However, C. difficile is known for its intrinsic resistance to lysozyme that might be associated with high level of deacetylation of its PG [START_REF] Ho | Clostridium difficile extracytoplasmic function sigma factor sigmaV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF]. Interestingly, using an antimicrobial disk assay realized on Pep-M plates, we observed a greater size of the zone of inhibition for the ∆prkC mutant (17 mm) when compared to the WT strain (7.25 mm) in presence of 800 μg of lysozyme (Fig 6B).

These results indicated that prkC deletion is involved in resistance to CAMPs and lysozyme.

prkC deletion affect motility, sedimentation and biofilm formation

To identify other phenotypes associated with cell envelop properties, we first tested motility on semi-solid BHI plates and observed a reduced motility for the ∆prkC mutant compared to the WT and complemented strains (Fig 7A). After 48 h, the motility of the WT strain is 1.35 mm +/-0.2 mm while the motility of the ∆prkC mutant is 0.8 mm +/-0.1 mm. We also observed that the ∆prkC mutant formed more aggregates at the bottom of the tube than the WT and complemented strains (Fig S7A). We also tested the ability of these strains to form biofilm after an exposure to compounds triggering an envelope stress such as DOC or polymixin B. In the presence of sub-inhibitory concentration of polymixin B (20 μg/ml) or DOC (0.01%), the ∆prkC mutant formed, respectively, 6-and 10-fold more biofilm than the WT and complemented strains after 24 h, respectively (Fig 7B andC). However, no difference in biofilm formation was observed without these compounds (data not shown). When we quantified the biofilm formed in presence of DOC at 24 h, 48 h and 72 h (Fig S7B ), we found that the ∆prkC mutant formed biofilm earlier than the WT strain (at 24 h) while more biofilm was produced by the WT strain than the ∆prkC mutant at 48 h. A previous work show that extracellular DNA (eDNA) is a major component of the matrix in DOC-induced biofilms [START_REF] Dubois | A microbiota-generated bile salt induces biofilm formation in Clostridium difficile[END_REF]. In both the ∆prkC mutant and the WT strain, DOC-induced biofilms were rapidly dispersed when treated with DNase (Fig S7C). Moreover, we detected eDNA in the extracellular matrix for the ∆prkC mutant at 24 h and 48 h but only at 48 h for the WT strain (Fig 7D).

Since eDNA is required for biofilm stability (29), the early release of eDNA detected in the ∆prkC mutant might be responsible for the premature biofilm formation. Thus, deletion of prkC seems to affect the motility, the ability of C. difficile to sediment and to form biofilm under conditions that induce cell envelop stress.

Comparison of the PG structure and composition between the WT strain and the ∆prkC mutant

Based on the changes in cell-shape and increased susceptibility to ß-lactams and lysozyme of the ∆prkC mutant, we hypothesized that these phenotypes could be due to modification of the PG structure or composition. However, we showed that the muropeptide profiles of the WT strain (black) and the ∆prkC mutant (red) are almost identical (Fig S8). Indeed, we found no difference in the abundance of dimers containing a 3-3 cross-link (peaks 9, 10, 11, 13, 14, 15 and 17) generated by L,D-transpeptidases that might be associated with the high level of intrinsic resistance of C. difficile to some ß-lactams [START_REF] Peltier | Clostridium difficile has an original peptidoglycan structure with a high level of Nacetylglucosamine deacetylation and mainly 3-3 cross-links[END_REF] Moreover, the ratio of 4-3 to 3-3 cross-linking was not affected in our conditions. In addition, the amount of deacetylated muropeptides (peaks 4, 7, 9, 10, 11, 13, 14, 15, 17, 18, 19, 21) (31), was similar in the WT strain and the ∆prkC mutant (Fig S8). As muropeptides deacetylation contribute to a high level of resistance to lysozyme in C. difficile [START_REF] Ho | Clostridium difficile extracytoplasmic function sigma factor sigmaV regulates lysozyme resistance and is necessary for pathogenesis in the hamster model of infection[END_REF], the increased sensitivity of the ∆prkC mutant to lysozyme cannot be explained by changes in the level of PG deacetylation (Fig S8). Our results suggested that susceptibility of the prkC mutant to some ß-lactams or lysozyme is not related to changes of its PG structure or composition.

Impact of the prkC deletion on the PSII

In firmicutes, the wall teichoic acids (WTA) are involved in cell division, maintenance of cell-shape and susceptibility to CAMPs or ß-lactam [START_REF] Brown | Wall teichoic acids of gram-positive bacteria[END_REF]. The 630∆erm strain of C. difficile has an atypical wall teichoic acid known as polysaccharide II (PSII) composed of hexaglycosylphosphate repeats [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF][START_REF] Ganeshapillai | Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units[END_REF]. To know if the phenotypes observed for the ∆prkC mutant were associated with changes in the abundance, localization and/or structure of the PSII, we first purified the PSII of the WT and ∆prkC mutant strains. Their structure was analyzed by NMR and showed that the 1 H NMR and 13 C HSQC spectra from the 630erm PSII were in good agreement with previous data [START_REF] Reid | Structural characterization of surface glycans from Clostridium difficile[END_REF][START_REF] Ganeshapillai | Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units[END_REF] and that no difference in the PSII structure was detected between the prkC mutant and the WT strain (Fig S9).

We also investigated a possible role for PrkC in controlling PSII localization. Using an immunoblot analysis with an antibody raised against PSII [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF], we observed that more PSII was shed in the supernatant during growth by the ∆prkC mutant when compared to the parental and complemented strains during growth (Fig 8A ). By contrast, we detected similar quantity of PSII in membrane fraction for all strains (Fig S10). Thus, this result suggests that the deletion of prkC leads to an enhanced release of PSII into the supernatant.

To precisely quantify the amount of PSII and PG in the cell wall of the WT and the ∆prkC mutant strains, we fractionated cells to recover the cell wall containing the PSII covalently linked to PG. We then separated these two compounds and quantified the amount of each product. Interestingly, we observed that the amount of PG purified from the ∆prkC mutant was lower compared to the WT strain, which represented 55 % of the quantity purified from

prkC deletion affects cell wall associated proteins

Several properties of the bacterial cell envelop can be partly attributed to proteins localized at the cell surface. We extracted proteins non-covalently anchored to the cell wall of the ∆prkC mutant and the WT strains ( 55) and compared their relative abundance by mass spectrometry (Table 2 andFig S11 ). A first family of proteins is the CWPs that contain a CWB2 motif responsible for their anchorage to the cell wall by interacting with PSII [START_REF] Kirk | Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics[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]. Indeed, we found that 27 CWP proteins with CWB2 (out of 29 in strain 630) including the Slayer protein, SlpA, were more abundant in the cell wall of the WT strain than in the ∆prkC mutant while a unique CWP protein, Cwp7, was more abundant in the ∆prkC mutant. Other non-covalently anchored proteins present of the cell surface (with SH3_3 or PG4 motifs) were also found in different amount in the WT strain and the ∆prkC mutant. Interestingly, some of them are involved in cell wall metabolism including L,D-transpeptidases, carboxypeptidases and putative cell wall hydrolases.

Virulence and colonization of the ∆prkC mutant in the hamster model

The different phenotypes of the prkC mutant suggest that PrkC can play a role during critical steps of the infectious cycle of C. difficile (4). To determine a possible role of PrkC in CDI, we compared the virulence of the WT strain and the ∆prkC mutant in the acute Golden Syrian hamster model of infection. Despite a trend towards a delay in the death of the hamsters infected with the prkC mutant, no significant difference was observed for the average time of post challenge survival between hamsters infected with the WT and the ∆prkC mutant strains (Fig 9A). When we monitored the daily level of gut colonization of the WT and the ∆prkC mutant, we found no significant difference in the average level of gut colonization the day the hamster died as determine by the average CFU per g of feces for each hamster (Fig 9B). However, a significant change in gut colonization was observed between the two strains 40 h post infection (Fig 9C). Despite the fact that the ∆prkC mutant has a delay in gut colonization, this delay has no significant effect on the virulence in this infection model.

Discussion

In this work, we showed that the prkC mutant of C. difficile has pleiotropic phenotypes such as an increased sensitivity to various antimicrobial compounds (CAMPs, lysozyme, DOC, ß-lactams), modifications in motility, cell aggregation and biofilm formation, and also changes in cell morphology and septum formation or localization. The increased sensitivity to several antimicrobial compounds detected for the C. difficile ∆prkC mutant has been observed in other firmicutes. The prkA mutant of L. monocytogenes is more sensitive to lysozyme and several CAMPs (58) while the ireK mutant of E. faecalis has an increased sensibility to bile and cholate [START_REF] Kristich | A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence[END_REF] but not to DOC as observed for the C. difficile ∆prkC mutant.

The inactivation of the PASTA-STKs in S. pneumoniae, S. pyogenes, E. faecalis, L. monocytogenes and S. aureus results in an increased susceptibility toward ß-lactams but the extent and pattern of the effects vary among species and strains [START_REF] Pensinger | Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets[END_REF][START_REF] Kristich | A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence[END_REF][START_REF] Pensinger | The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence[END_REF][START_REF] Dias | The highly conserved serine threonine kinase StkP of Streptococcus pneumoniae contributes to penicillin susceptibility independently from genes encoding penicillin-binding proteins[END_REF]. These specific patterns of sensitivity observed for the PASTA-STK mutant for each firmicute might be associated with differences in the targets phosphorylated by the PASTA-STK, in the penicillin-binding proteins (PBPs) and in their affinity for the different ß-lactams. As observed in B. subtilis and S. aureus [START_REF] Pensinger | Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets[END_REF][START_REF] Madec | Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes[END_REF], the PrkC kinase of C. difficile controls biofilm formation under cell-membrane stresses conditions. Several factors probably contribute to the increased biofilm formation in the ∆prkC mutant in the presence of DOC or polymixin B. This may include a decreased motility, an increased sedimentation and changes in the amount of CWPs that modify adhesive properties of the cell surface. For example, the Cwp84 protein, which is less abundant in the ∆prkC mutant is known to negatively control biofilm formation [START_REF] Pantaleon | The Clostridium difficile Protease Cwp84 Modulates both Biofilm Formation and Cell-Surface Properties[END_REF]. In addition, premature autolysis by the ∆prkC mutant probably releases eDNA earlier in presence of DOC resulting in the increased biofilm formation observed at 24 h for the mutant [START_REF] Dubois | A microbiota-generated bile salt induces biofilm formation in Clostridium difficile[END_REF]. By controlling cell lysis, PrkC seems able to affect biofilm formation in response to cell membrane stresses.

Changes in the homeostasis and the integrity of the cell envelop might explain most of the phenotypes of the C. difficile ∆prkC mutant including modification of cell shape that is determined by the orderly processing and assembly of each cell wall component [START_REF] Brown | Wall teichoic acids of gram-positive bacteria[END_REF][START_REF] Siegel | Biogenesis of the Gram-positive bacterial cell envelope[END_REF][START_REF] Typas | From the regulation of peptidoglycan synthesis to bacterial growth and morphology[END_REF].

The absence of PrkC probably modifies the abundance, composition and/or structure of component(s) of the cell envelop, including the PG, the glycopolymers (PSII and/or LTA-PSIII) and/or cell-wall associated proteins [START_REF] Kirk | Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics[END_REF]. In other Gram-positive bacteria, PASTA-STK phosphorylates enzymes involved in PG and/or teichoic acid synthesis, modification, assembly and/or turnover. This includes enzymes of the Glm pathway, Mur enzymes, MviM, a flippase involved in the transport of the lipid II anchored compounds across the membrane, a penicillin-binding protein, LTA synthetases but also transcriptional regulators such as WalR-WalK and GraR that control cell wall metabolism [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF][START_REF] Pensinger | Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets[END_REF][START_REF] Pompeo | Investigation of the phosphorylation of Bacillus subtilis LTA synthases by the serine/threonine kinase PrkC[END_REF]. In C. difficile, we did not detect a transcriptional effect of the prkC deletion on genes encoding enzymes involved in the synthesis of envelop components (E. Cuenot, unpublished data). These results strongly suggest that PrkC does not act by modifying the activity of a transcriptional regulator but rather by phosphorylating one or several protein(s) directly or indirectly controlling the synthesis, assembly or turnover of at least one component of the envelop.

Teichoic acids play a role in protecting bacteria from stressful conditions by modifying the properties of the cell surface. Changes in teichoic acids resulted in phenotypes similar to those we observed in the ∆prkC mutant. This include increased ß-lactam susceptibility, autolysis, modification of cell morphology and defect in septa positioning and numbers [START_REF] Brown | Wall teichoic acids of gram-positive bacteria[END_REF][START_REF] Percy | Lipoteichoic acid synthesis and function in grampositive bacteria[END_REF]. In C. difficile, the biosynthesis of the PSII, its properties as well as the phenotypes associated with defect in PSII are poorly studied [START_REF] Reid | Structural characterization of surface glycans from Clostridium difficile[END_REF][START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF]. Furthermore, less information is available on atypical LTA [START_REF] Reid | Structural characterization of surface glycans from Clostridium difficile[END_REF]. However, we noted that the ∆prkC mutant shares common phenotypes with the lcpB mutant encoding a protein involved in tethering PSII (34) that includes elongated cell, presence of multiple septa and increased ability to form biofilms [START_REF] Chu | A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence[END_REF]. While the structure of PSII is not altered in the ∆prkC mutant, we observed a slightly reduction in the amount of PSII anchored to the cell wall and an increased quantity in PSII released in the supernatant of the ∆prkC mutant. These changes in PSII localization might be caused by the dysregulation of PSII synthesis/anchorage or the production/assembly of another cell envelope component such as PG in the absence of PrkC. Indeed, anchoring of the PSII to PG is probably affected by the reduced amount of PG detected in the ∆prkC mutant.

As observed for L. monocytogenes and E. faecalis, C. difficile is inherently resistant to cephalosporin and PASTA-STK inactivation leads to a robust increase in sensitivity towards these antibiotics that target PG synthesis by inactivating PBPs [START_REF] Kristich | A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence[END_REF][START_REF] Pensinger | Selective pharmacologic inhibition of a PASTA kinase increases Listeria monocytogenes susceptibility to beta-lactam antibiotics[END_REF]. In C. difficile, we showed that the kinase activity of PrkC is required for the high level of resistance to cephalosporin. The sensitivity of the ∆prkC mutant to teicoplanin, an antibiotic that affects transglycosylation by PBPs and to bacitracin that interferes with PG synthesis through its role on lipid II recycling also suggests a possible role for this kinase in controlling PG metabolism [START_REF] Eggert | Genetic basis for activity differences between vancomycin and glycolipid derivatives of vancomycin[END_REF]. Other phenotypes such as lysozyme resistance or autolysis are also related to PG struc-ture or metabolism. However, we failed to detect any modification in the composition of PG, in reticulation of PG, 3-3 to 4-3 cross-link ratio or PG deacetylation under our experimental conditions. Changes to these would have offer, to a certain extent, an explanation for the increased sensitivity to cephalosporin and lysozyme. Nevertheless, it is possible that localized and/or subtle changes in PG composition occur at the septum, which is where PrkC is localized during growth. These changes would be difficult to detect. The main difference between the ∆prkC and the WT is a decrease in the total amount of PG. This might be linked to a reduced size of the glycan chains formed. It is interesting to note that proteins potentially involved in PG metabolism are also detected in different amount in the cell wall of the WT and ∆prkC mutant strains (Table 2). All these results suggest that PrkC has an effect directly or indirectly on PG synthesis or turnover in C. difficile. In L. monocytogenes, it has been suggested that the PASTA kinase might specifically regulate PBPs resulting in cephalosporin resistance [START_REF] Pensinger | The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence[END_REF]. In Enterococcus faecium, mutants in class A PBPs are sensitive to cephalosporins and suppressor mutations that restore cephalosporin resistance are found in ireK and ireP encoding a STK and a STP, respectively [START_REF] Desbonnet | Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium[END_REF]. In E. faecalis, IreB negatively controls cephalosporin resistance and IreB is a small protein of unknown function and the only substrate of IreK kinase identified to date [START_REF] Hall | IreB, a Ser/Thr kinase substrate, influences antimicrobial resistance in Enterococcus faecalis[END_REF]. However, the molecular mechanisms linking IreB, IreK and maybe PBPs that could explain their role in the control of cephalosporin resistance remain unknown. Interestingly, CD1283, the IreB-like protein of C. difficile, contains one threonine (T7) and this residue is phosphorylated in IreB of E. faecalis.

CD1283 might contribute to the regulatory pathway downstream of PrkC. In addition, the PrkC-mediated phosphorylation of proteins controlling cell division and the synthesis of cell envelop components is another interesting hypothesis. Indeed, PrkC is localized at the septum and the deletion of the prkC gene affects C. difficile cell morphology with elongated cells, abnormal septum localization and defect in cell separation. In S. pneumoniae and other streptococci, ∆stkP mutants have longer cells than the WT and a modified shape and exhibits cell division and separation defects [START_REF] Fleurie | Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae[END_REF]. Several proteins involved in cell division such as DivIVA, MapZ, GspB or FtsZ are phosphorylated by the PASTA-STKs in B. subtilis, S. pneumoniae or S. aureus [START_REF] Manuse | Role of eukaryoticlike serine/threonine kinases in bacterial cell division and morphogenesis[END_REF][START_REF] Pensinger | Do Shoot the Messenger: PASTA Kinases as Virulence Determinants and Antibiotic Targets[END_REF][START_REF] Fleurie | Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae[END_REF]. In S. pneumoniae, phosphorylation of DivIVA controls cell shape and the localization of PG synthesis machinery required for cell elongation and cell constriction [START_REF] Fleurie | Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae[END_REF]. The phosphorylation by PrkC of an orthologous of one of these proteins, especially DivIVA, could explain several cell morphology associated phenotypes observed for the prkC mutant. Altogether, our results indicate that the PASTA-STK of C. difficile, PrkC, is probably involved in the control of the envelop biogenesis and/or cell division.

Finally, the virulence of the ∆prkC mutant is similar to that of the WT strain while a colonization delay of the hamster gut is observed for the mutant. This can be explained by a global increased in sensitivity of the ∆prkC mutant to antimicrobial compounds and the possible changes in its cell envelop properties. Our results highlight the involvement of PrkC in controlling several processes corresponding to critical steps of CDI including resistance to DOC, known to inhibit growth of vegetative cells in the gastrointestinal tract (4), to lysozyme, a critical component of the innate immune system and to CAMPs produced by the microbiota and/or by the host (3) that are compounds present in the hamster model. Furthermore, dissecting the role of PrkC in controlling the resistance to antibiotics promoting CDI such as cephalosporins (1) could pave the way to new strategies for the prevention of these infections.

Acknowledgments.

We thank to Johann Peltier for helpful discussions and experimental advices, Gayatri Vedantam for the gift of the antibody raised against PSII, Jost Eninga for access to the fluorescent microscope, Nigel Minton for the genetic tools, Nicolas Kint for its help for the construction of the ∆prkC mutant and Sandrine Poncet for helpful advices. This work was funded by the Institut Pasteur, the University Paris 7, the ITN Marie Curie, Clospore (H2020-MSCA-ITN-2014 642068) and the ANR DifKin (ANR-17-CE15-0018-01). EC and TGG are the recipient of a ITN Marie Curie and an ANR fellowships, respectively. Cell-wall binding protein, glucosaminidase domain 3.9 4.4 +

CD0844 cwp25

Cell-wall binding protein 3.9 5.9

+ CD3192 cwp21
Cell-wall binding protein 3.9 6.8 CD2789 cwp66

Cell-wall binding protein The conserved lysine residue (K39) within the ATP-binding P loop of the kinase that is required for phosphotransfer is indicated. B. Localization of the PrkC-HA tagged protein. Cells expressing prkC fused to HA were grown in the presence of 15 ng/ml of ATc, and harvested during exponential growth. Samples were fractionated into membrane (Mb) and cytoplasm (Cy) fractions. Protein fractions were analyzed by western blot using an antibody raised against HA. C. Localization of the SNAP-PrkC fusion during growth. The SNAP-PrkC protein was produced during exponential growth phase in the presence of 50 ng/ml ATc. After labeling with the TMR-star substrate, PrkC-SNAP localization was analyzed by fluorescence microscopy. AF (autofluorescence). The scale bar represents 5 μm. D. Western blot performed after fractionation in a soluble fraction (1) and an insoluble fraction (2) obtained from exponential phase cultures of the WT, the ∆prkC mutant, the complemented strain and the ∆prkC mutant carrying pDIA6103-prkC-K39→A (K→A). An α-P-Thr antibody was used to detect phosphorylated threonine. We indicated by red arrows the bands detected in the WT strain that disappeared in the ∆prkC mutant. Figure 2. Genetic organization of the prkC locus A. Schematic representation of the gene cluster present upstream of prkC. The locus between CD2590-dapF and prkC includes genes encoding proteins involved in translation, transcription, DNA replication, metabolism and membrane proteins. The genome-wide TSS mapping [START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF] indicated the presence of a promoter upstream of dapF. The extended -10 box and the TSS are indicated in bold. B. PCR realized with primers annealing in prkC and stp (IMV936 and IMV908, line 1 and 2) or stp and rlmN (IMV935 and IMV907, line 3 and 4) either on RNA extracted from 630∆erm (line 1 and 3) or on cDNA synthesized by reverse transcription from the same RNA using primer IMV843 (line 2 and 4). Smart Ladder (200-10,000 bp). B. Cell size distribution for the WT, the ∆prkC mutant and the complemented (Comp) strains. The measurement was done on cells labeled with FM4-64 and DAPI. Means and errors of the means were calculated after the measurement of at least 600 cells for each strain. The analysis was performed with the software Imaje J. C. Presence of aberrant septation and anucleated minicells in ΔprkC mutant cells. Fluorescence microscopy of cells staining with FM4-64 and DAPI revealed the presence of aberrant septum (yellow arrows) and anucleate minicells (white arrows) in the ∆prkC mutant. Scale bars represent 5 μm. Resistance to SDS stress of the ∆prkC mutant, the 630∆erm (WT) and the complement strains (Comp) was tested on BHI plates containing 0.006 % of SDS. This experiment was performed in triplicate and this plate is representative of the results obtained. B. Growth of the WT strain (black circle), the ∆prkC mutant (white circle) and the complemented strain (white square) in 24-well microplates containing TY medium in the presence of 0.03% DOC. A growth curve without DOC is presented in Fig S3 . 4 independent cultures were done. C. Autolysis of the WT (black circle), ∆prkC (white circle) and complemented (black square) strains in the presence of 0.01% Triton X-100. The OD600nm of the samples incubated at 37°C was determined every 5 min until complete cell lysis was reached. 4 independent experiments were done. Figure 6. Sensitivity of the ∆prkC mutant to antibiotics targeting cell-wall and to lysozyme. A. Sensitivity to antibiotics targeting the cell wall. Histograms representing the diameters of growth inhibition area after 24 h of incubation on BHI plates for the 630∆erm strain pDIA6103 (WT, black), the ∆prkC mutant pDIA6103 (medium grey), the complemented strain (Comp, dark grey) and the ∆prkC mutant expressing PrkC-K39A (pale grey). We used antibiogram disks containing Ticarcillin 75 μg, Amoxicillin 25 μg, Imipenem 10 μg, Ceftazidime 30 μg, Cefepime 30 μg or Erythromycin 15 μg. Cefoxitin was tested at 100 μg. The results presented correspond to 7 experiments (Ticarcillin, Amoxicillin, Imipenem) or 4 experiments for cephalosporins. Data were analyzed by t test. * indicates P<0.05. B. Sensitivity to lysozyme was determined on Pep-M plates. 800 μg of lysozyme was added to a 6-mm disk. Histograms representing the diameter of growth inhibition measured for the 630∆erm pDIA6103 (WT, black), the ∆prkC mutant pDIA6103 (medium grey), and the complemented strain (Comp, dark grey). The experiment was performed in quadruplicate. Data were analyzed by t test. * indicates P<0.05.

  S1). A DNA fragment obtained by overlapping PCR was digested by XhoI and BamHI and then ligated into pMTLSC7315∆MCS, giving pDIA6401. E. coli HB101 (RP4) containing pDIA6401 was mated with C. difficile 630∆erm. Transconjugants were selected on BHI-Cfx-Tm plates. Colonies were restreaked onto the same medium to identify faster growing single-crossover integrants that were then streaked onto CDMM supplemented with 50 μg/ml fluorocytosine (FC) to select for second cross-over events. FC R Tm S clones that have lost pDIA6401 were analyzed by PCR using IMV867 and IMV868 to distinguish between wild-type and ∆prkC clones (FigS1). For complementation, the prkC gene (positions -22 to +2205 from the translational start site) was amplified using primers IMV879 and IMV880. The PCR fragment was cloned between the StuI and BamHI sites of pDIA6103[START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF] to produce pDIA6413. To construct a plasmid expressing PrkC-HA (pDIA6713), an reverse PCR was performed using pDIA6413 as template and primers EC01 and EC02. The same strategy was used to introduce a point mutation into the prkC gene [lysine at position 39 replaced by an alanine (K39→A)]. The resulting plasmid, pDIA6714, was then used to construct pDIA6716 expressing HA-tagged prkC(K39→A). These plasmids were transferred into C. difficile strains by conjugation (Table S1).

  PASTA-STK of C. difficile CD2578 (PrkC) from C. difficile contains a cytosolic N-terminal kinase domain and an extracellular motif containing repeats of a PASTA that are separated by a trans-membrane segment (Fig 1A). Based on its predicted amino acid sequence, the kinase domain of PrkC has a high level of sequence identity with that of B. subtilis (PrkC), S. aureus (Stk1), S. pneumoniae (StkP), Enteroccoccus faecalis (IreK) and Mycobacterium tuberculosis (PknB) (43 to 50%). The conserved lysine within the ATP-binding P loop of the kinase domain is present and corresponds to position 39 in PrkC (Fig 1A). This residue is essential for the phosphotransfer in other STKs (41). As observed for the PASTA-STKs in other firmicutes, a trans-membrane segment is predicted from amino acid 375 to 397 (42, 43). The extracellular sequence of PrkC contains 2 PASTA motifs (Fig 1A) while up to 7 PASTA motifs are typically present in STKs of other firmicutes (17). Interestingly, C. difficile contains a very atypical Ser-Gly-Asn (SGN) rich region of 100 amino acids at the C-terminal part of the protein (Fig 1A). This SGN-rich region is absent from PASTA-STKs of other firmicutes. Interestingly, SGN-rich regions are found in some cell wall anchored proteins of bacilli and in CwpV, a surface associated protein in C. difficile but their function have yet to be characterized.

  4 μm, respectively (Fig 3B). We detected elongated cells (Fig 3A, blue arrows) and cells with several septa and undivided (Fig 3A, green arrows). Interestingly, we observed septation defects for the ∆prkC mutant (Fig 3A, white square, Fig 3C and Fig S5B). Our analysis revealed that 4% of the cells had abnormal septation (Fig 3C and S5B, yellow arrows) and that 1% of the cells had adjacent septa which created mini-cell lacking DAPI-stained DNA (Fig 3C and Fig S5B, white arrows). We then analyzed by TEM the cell morphology and the structure of the septa for the WT, the ∆prkC mutant and the complemented strains. Cells with normal septation were observed for the WT strain, complemented strain and ∆prkC mutant (Fig 4A) while some cells of the ∆prkC mutant displayed aberrant septation with the presence of multiple and adjacent septa (Fig 4B). Mini-cells attached to each other were also observed (Fig 4B left) and these probably arise from the formation of several septa in close proximity (Fig 4B, right, red arrows). These results indicated that deletion of the prkC gene in C. difficile affects bacterial cell morphology and septum formation.

  or containing a 4-3 crosslink (peaks 18, 19, 21) catalyzed by D,D-transpeptidases, a target of ß-lactam (Fig S8).

  the WT strain (Fig 8B). Similarly, but to a lesser extent, a reduction of 33 % in the quantity of PSII was observed for the ∆prkC mutant (Fig 8C). It is possible that the reduction in PG for the ∆prkC mutant could interfere with the localization of PSII and could explain the increase shedding of PSII in the supernatant (Fig 8A).

Figure 1 .

 1 Figure Legend Figure 1. Organization, localization and kinase activity of the PrkC protein. A. Organization of the domains of C. difficile CD2578-PrkC. PrkC contains a cytoplasmic kinase domain in N-terminal part (brown), a trans-membrane (TM) segment, two PASTA domains (green) and an atypical SGN (Ser, Gly, Asn) rich domain in C-terminal part (pink).The conserved lysine residue (K39) within the ATP-binding P loop of the kinase that is required for phosphotransfer is indicated. B. Localization of the PrkC-HA tagged protein. Cells expressing prkC fused to HA were grown in the presence of 15 ng/ml of ATc, and harvested during exponential growth. Samples were fractionated into membrane (Mb) and cytoplasm (Cy) fractions. Protein fractions were analyzed by western blot using an antibody raised against HA. C. Localization of the SNAP-PrkC fusion during growth. The SNAP-PrkC protein was produced during exponential growth phase in the presence of 50 ng/ml ATc. After labeling with the TMR-star substrate, PrkC-SNAP localization was analyzed by fluorescence microscopy. AF (autofluorescence). The scale bar represents 5 μm. D. Western blot performed after fractionation in a soluble fraction (1) and an insoluble fraction (2) obtained from exponential phase cultures of the WT, the ∆prkC mutant, the complemented strain and the ∆prkC mutant carrying pDIA6103-prkC-K39→A (K→A). An α-P-Thr antibody was used to detect phosphorylated threonine. We indicated by red arrows the bands detected in the WT strain that disappeared in the ∆prkC mutant.Figure 2. Genetic organization of the prkC locusA. Schematic representation of the gene cluster present upstream of prkC. The locus between CD2590-dapF and prkC includes genes encoding proteins involved in translation, transcription, DNA replication, metabolism and membrane proteins. The genome-wide TSS mapping[START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF] indicated the presence of a promoter upstream of dapF. The extended -10 box and the TSS are indicated in bold. B. PCR realized with primers annealing in prkC and stp (IMV936 and IMV908, line 1 and 2) or stp and rlmN (IMV935 and IMV907, line 3 and 4) either on RNA extracted from 630∆erm (line 1 and 3) or on cDNA synthesized by reverse transcription from the same RNA using primer IMV843 (line 2 and 4). Smart Ladder (200-10,000 bp). Figure3. Morphology of the ∆prkC mutant A. Fluorescence microscopy was carried out on the WT, the ∆prkC mutant and the complemented (Comp) strains. Membranes and DNA were visualized with FM4-64 (red) and DAPI (blue), respectively. A blue arrow showed an elongated cell and green arrows non-separated cells. The white square indicates the presence of a septum with an aberrant structure. Scale bars represent 5 μm.

Figure 3 .

 3 Figure Legend Figure 1. Organization, localization and kinase activity of the PrkC protein. A. Organization of the domains of C. difficile CD2578-PrkC. PrkC contains a cytoplasmic kinase domain in N-terminal part (brown), a trans-membrane (TM) segment, two PASTA domains (green) and an atypical SGN (Ser, Gly, Asn) rich domain in C-terminal part (pink).The conserved lysine residue (K39) within the ATP-binding P loop of the kinase that is required for phosphotransfer is indicated. B. Localization of the PrkC-HA tagged protein. Cells expressing prkC fused to HA were grown in the presence of 15 ng/ml of ATc, and harvested during exponential growth. Samples were fractionated into membrane (Mb) and cytoplasm (Cy) fractions. Protein fractions were analyzed by western blot using an antibody raised against HA. C. Localization of the SNAP-PrkC fusion during growth. The SNAP-PrkC protein was produced during exponential growth phase in the presence of 50 ng/ml ATc. After labeling with the TMR-star substrate, PrkC-SNAP localization was analyzed by fluorescence microscopy. AF (autofluorescence). The scale bar represents 5 μm. D. Western blot performed after fractionation in a soluble fraction (1) and an insoluble fraction (2) obtained from exponential phase cultures of the WT, the ∆prkC mutant, the complemented strain and the ∆prkC mutant carrying pDIA6103-prkC-K39→A (K→A). An α-P-Thr antibody was used to detect phosphorylated threonine. We indicated by red arrows the bands detected in the WT strain that disappeared in the ∆prkC mutant.Figure 2. Genetic organization of the prkC locusA. Schematic representation of the gene cluster present upstream of prkC. The locus between CD2590-dapF and prkC includes genes encoding proteins involved in translation, transcription, DNA replication, metabolism and membrane proteins. The genome-wide TSS mapping[START_REF] Soutourina | Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile[END_REF] indicated the presence of a promoter upstream of dapF. The extended -10 box and the TSS are indicated in bold. B. PCR realized with primers annealing in prkC and stp (IMV936 and IMV908, line 1 and 2) or stp and rlmN (IMV935 and IMV907, line 3 and 4) either on RNA extracted from 630∆erm (line 1 and 3) or on cDNA synthesized by reverse transcription from the same RNA using primer IMV843 (line 2 and 4). Smart Ladder (200-10,000 bp). Figure3. Morphology of the ∆prkC mutant A. Fluorescence microscopy was carried out on the WT, the ∆prkC mutant and the complemented (Comp) strains. Membranes and DNA were visualized with FM4-64 (red) and DAPI (blue), respectively. A blue arrow showed an elongated cell and green arrows non-separated cells. The white square indicates the presence of a septum with an aberrant structure. Scale bars represent 5 μm.

Figure 4 .

 4 Presence of abnormal septa in the ∆prkC mutant cells A. TEM pictures showing normal septal structure in the 630∆erm (WT), the ∆prkC mutant and the complemented (Comp) strains. Scale bars represent 100 nm. B. TEM pictures showing aberrant septal structure in the ∆prkC mutant cells. In the right panel, we can distinguish the beginning of the synthesis of two septa (white arrows) has begun near an apparent normal septum. Scale bars represent 200 nm.

Figure 5 .

 5 prkC deletion increases sensitivity to detergents and autolysis A.

Figure 7 .

 7 Sedimentation, biofilm formation and motility of the ∆prkC mutant A. Motility test on 0.3% agar BHI plates. The plates shown are representatives of 3 independent tests. B and C Mean values of the OD570nm measured after crystal violet staining of the mass of biofilm obtained after 24 h in presence of 20 μM polymixin B (B) or 0.01% DOC (C). Error

Table 1 . MIC of the strain 630∆erm and of the ∆prkC mutant for antibiotics targeting cell wall and CAMPS

 1 MICs for antibiotics were determined using E-test with the exception of cefotaxime.The MICs for cefotaxime and antimicrobial peptides were determined by the method of dilution.

	Compound tested	MIC (µg/ml)	Median-fold
		630∆erm	∆prkC	sensitization
	Antibiotics targeting cell wall
	Vancomycin	1.5	0.75	2
	Teicoplanin	0.19	<0.016	>12
	Amoxicillin	4	1	4
	Imipenem	6	1.3	4
	Cefoxitine	>256	44	>6
	Ceftazidime	60	3	20
	Cefepime	70	4	17.5
	Cefotaxime	128	16	8
	Antimicrobial peptides
	Polymixin B	340	55	6
	Bacitracin	550	25	22
	Nisin	140	30	4.5

Table 2 . Change in cell wall associated proteins in C. difficile between the 630∆erm 717 strain and the ∆prkC mutant.

 2

	718
	Gen ID	name	function	Fold-change	-Log	Detected in
				630∆erm/	(P-value)	surface
				∆prkC*		proteome
	Cell-wall associated proteins CWB2 motif
	Down in ∆prkC
	CD1233	cwp26	Cell wall binding protein of skin element 31.3	2.7
	CD1469	cwp20	Cell-wall penicillin-binding protein	6.7	6.5
	CD2518	cwp29	Cell-wall binding protein	6.4	4.6
	CD2795	cwp11	Cell-wall binding protein	6.1	6.5
	CD0440	cwp27	Cell-wall binding protein	5.1	4.3
	CD2735 # cwp14	Cell-wall binding protein (also SH3)	4.8	5.2
	CD2798	cwp9	Cell-wall binding protein	4.7	4.5
	CD2767	cwp19	Cell-wall binding protein, autolysin	4.6	5.9
	CD1803	cwp23	Cell-wall binding protein	4.4	4.9
	CD2786	cwp5	Cell-wall binding protein	4.4	5.4
	CD1751	cwp13	Cell-wall binding protein, protease	4.2	5.4
	CD2787	cwp84	Cell-wall binding protein, protease	4.0	5.4	+
	CD2193	cwp24

bars show standard deviation of three independent experiments performed in triplicate. Data were analyzed by t test. P values were 0.031 and 0.0045 for polymixin B (B) and DOC (C), respectively. On the picture, crystal violet staining revealing the amount of biofilm formed after 24 h corresponding to the values represented above. D. Detection of e-DNA in the matrix of 24 h and 48 h DOC-induced biofilms formed by the WT strain and the ∆prkC mutant. The experience was done in triplicate. We presented a gel representative of the results obtained.