Proteomic and genomic characterization of highly infectious 1

Clostridium difficile 630 spores 2

3

Trevor D. Lawley1*, Nicholas J. Croucher2, Lu Yu3, Simon Clare1, 4

Mohammed Sebaihia2, David Goulding1, Derek J. Pickard1, Julian Parkhill2, 5

Jyoti Choudhary3 and Gordon Dougan1 6

7

1Microbial Pathogenesis Laboratory, 2Pathogen Genomics, 3Proteomics 8

Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, 9

Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK. 10

11

12 *corresponding author tl2@sanger.ac.uk 13

14

Trevor D. Lawley 15

Microbial Pathogenesis Laboratory 16

Wellcome Trust Sanger Institute 17

Hinxton, Cambridgeshire, UK 18

CB10 1SA 19

Phone 01223 495 391 20 Fax 01223 495 239 21 22 Running title 23 Clostridium difficile spores 24 25 26 27 28 29 30 31 32 33 34

35 36

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00597-09 JB Accepts, published online ahead of print on 19 June 2009

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ABSTRACT 37

Clostridium difficile, a major cause of antibiotic-associated diarrhea, produces highly 38

resistant spores that contaminate hospital environments and facilitate efficient disease 39

transmission. We purified C. difficile spores using a novel method and show that they 40

exhibit significant resistance to harsh physical or chemical treatments and are also highly 41

infectious, with <7 environmental spores per cm2 reproducibly establishing a persistent 42

infection in exposed mice. Mass spectrometric analysis identified ~336 spore-associated 43

polypeptides, with a significant proportion linked to translation, sporulation/germination 44

and protein stabilization/degradation. In addition, proteins from several distinct metabolic 45

pathways associated with energy production were identified. Comparison of the C. 46

difficile spore proteome to other clostridial species defined 88 proteins as the clostridial 47

spore “core” and 29 proteins as C. difficile spore-specific, including proteins that could 48

contribute to spore-host interactions. Thus, our results provide the first molecular 49

definition of C. difficile spores opening up new opportunities for the development of 50

diagnostic and therapeutic approaches. 51

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INTRODUCTION 60

Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that 61

can asymptomatically colonize the intestinal tract of humans and other mammals (3, 30, 62

39). Antibiotic treatment can result in C. difficile overgrowth and can lead to clinical 63

disease ranging from diarrhea to life threatening pseudomembraneous colitis, particularly 64

in immunocompromised hosts (2, 4, 7). In recent years, C. difficile has emerged as the 65

major cause of nosocomial antibiotic-induced diarrhea that is frequently associated with 66

outbreaks (21, 22). A contributing factor is that C. difficile can be highly infectious and 67

difficult to contain, especially when susceptible patients are present in the same hospital 68

setting (13). 69

Person-to-person transmission of C. difficile is associated with the excretion of 70

highly resistant spores in the feces of infected patients, creating an environmental 71

reservoir that can confound many infection control measures (29, 44). Bacterial spores, 72

which are metabolically dormant cells that are formed following asymmetric cell 73

division, are normally composed of thick concentric external layers, the spore coat and 74

cortex, that protects the internal cytoplasm (15, 42). Upon germination, spores lose their 75

protective external layers and resume vegetative growth (24, 27, 36). Bacillus and most 76

Clostridia spores germinate in response to amino acids, carbohydrates or potassium ions 77

(24, 36). In contrast, C. difficile spores show an increased level of germination in 78

response to cholate-derivatives found in bile (40, 41). Thus, spores are well adapted for 79

survival and dispersal in a wide range of environmental conditions but will germinate in 80

the presence of specific molecular signals (24, 36). 81

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Whilst the spores of a number of Bacilli, such as Bacillus subtilis and B. anthracis 82

and other Clostridia species such as C. perfringens (15, 20) have been well characterized, 83

research on C. difficile spores has been relatively limited. A greater understanding of C. 84

difficile spore biology could be exploited to rationalize disinfection regimes, molecular 85

diagnostics and the development of targeted treatments such as vaccines. Here we 86

describe a novel method to isolate highly purified C. difficile spores that maintain their 87

resistance and infectious characteristics, thus providing a unique opportunity to study C. 88

difficile spores in the absence of vegetative cells. A thorough proteomic and genomic 89

analysis of the spore provides novel insight into the unique composition and predictive 90

biological properties of C. difficile spores that should underpin future research into this 91

high profile but poorly understood pathogen. 92

93

MATERIALS AND METHODS 94

Strains and growth conditions 95

Clostridium difficile strain 630 (tcdA+tcdB

+; epidemic strain isolated in 1985 from 96

Zurich, CH; 012 ribotype) was routinely grown in BHI broth or on BHI agar plates 97

(Oxoid Limited, Basingstoke, UK) or in Wilson’s broth (45) at 37°C under anaerobic 98

conditions (Mini-Mac 250, Don Whitley, UK). To enumerate total C. difficile, samples 99

from cultures were removed from the anaerobic cabinet, serially diluted in PBS, plated on 100

BHI + 0.5% taurocholate agar plates and immediately returned to the anaerobic cabinet. 101

To enumerate spores, 0.1 ml samples were mixed with 0.1 ml of 100% ethanol for 1 hour 102

at room temperature to kill vegetative cells (5). Samples were pelleted and washed twice 103

in PBS before re-suspending in 0.1 ml of PBS. Spores were enumerated by serially 104

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diluting in PBS, plated on BHI agar plates containing 0.5% taurocholate. C. difficile was 105

grown for 24-48 hours at 37°C under anaerobic conditions. 106

107

Spore purification 108

Prior to spore purification, C. difficile was grown statically in 500 ml of Wilson’s 109

broth (45) under anaerobic conditions for 7-10 days. Cultures were pelleted by 110

centrifugation at 10 000 rpm for 15 minutes in a Sorvall RC 5C Plus centrifuge using a 111

SLA 3000 rotor and re-suspended in 35 ml of sterile water. Samples were washed in 112

water 4-6 times during which the supernatent became clear. Samples were then pelleted 113

and re-suspended in 30 ml of PBS prior to sonicating for 90 seconds at 35% amplitude in 114

a VibraCell sonicator (Sonics, Newton, USA) with a tapered probe (Model CV33). Then 115

3 ml of 10% Sarkosyl NL30 (VWR International Ltd., UK) was added to the samples 116

before incubation at 37°C with agitation for 1 hour. Samples were then pelleted with 117

centrifugation at 4 000 rpm for 10 minutes in a Sorvall Legend RT centrifuge. Pellets 118

were re-suspended in 10 ml of PBS containing 0.125 M Tris buffer (pH 8) and 10 mg/ml 119

lysozyme (Roche, Mannheim, Germany) and incubated overnight (~16 hours) at 37°C 120

with agitation. Samples were then sonicated as described previously and 1% Sarkosyl 121

was then added to the samples prior to incubation for 1 hour at 37°C with agitation. 122

Samples were then layered onto a 50% sucrose and centrifuged for 20 minutes at 4 000 123

rpm in a Sorvall Legend RT centrifuge. Pellets were re-suspended in 2 ml of PBS 124

containing 200mM EDTA, 300 ng/ml Proteinase K (recombinant PCR grade; Roche, 125

Mannheim, Germany) and 1% Sarkosyl NL 30 (VWR, Poole, UK) and incubated for 1-2 126

hours with shaking at 37°C until the samples cleared. Samples were then layered onto 127

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50% sucrose and centrifuged as described previously. Pellets were re-suspended in water 128

and washed twice prior to re-suspending in sterile water. Spores were stored at 4ºC. 129

Typically, the original 500 ml culture contained 5x109 to 1x1010 of viable C. difficile, of 130

which ~108-109 were ethanol resistant spores. The purification protocol typically 131

recovered >90% of the input spores. 132

133

Endospore staining 134

Staining of C. difficile cultures or pure spores was performed according to the 135

directions of the Schaeffer and Fulton Spore Stain Kit (Sigma, UK). Stains were viewed 136

on a Zeiss Axiovert 200m microscope equipped with a CCD camera. 137

138

Spore inactivation and germination assays 139

The concentration of the spore preparation was determined by visual enumeration 140

of serial dilutions using a hemocytometer. The spore preparations were devoid of 141

vegetative cells so the spores easily are recognized by their characteristic phase bright 142

appearance (23). 143

Spore germination and outgrowth. 144

Pure spores were serially diluted and plated on BHI agar plates (germination 145

background control). Preliminary experiments demonstrated that C. difficile spores 146

germinated equally well on BHI, Wilson’s and Brazier’s agar plates. For experiments, 147

the BHI agar plates were supplemented with either cholate, taurocholate or glycocholate 148

at a concentration of 0.1%. C. difficile was grown for 48 hours before counting. 149

Heat and Chemical Inactivation 150

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To test for heat resistance, pure spores were placed in water in a total volume of 151

0.5 ml. Samples were placed in a heating block set to 60°C or 70°C or left at room 152

temperature. At 0.5, 3 and 24 hours, samples were removed and enumerated by culturing 153

as described above. To test for chemical resistance, pure spores were placed in either 154

70% ethanol (VWR, UK) or 1% Virkon (strong oxidizing agent with the active 155

compound potassium peroxymonosulfate)(Antec International, Suffolk, UK) in a total 156

volume of 0.5 ml for 20 minutes. Treated spores were pelleted with centrifugation, 157

washed in sterile water before enumeration as described above. 158

159

Electron microscopy 160

Pelleted spores were resuspended in freshly prepared primary fixative that 161

contained 2% paraformaldehyde and 2% glutaaldehyde in 0.1M sodium cacodylate buffer 162

(pH 7.42) with added 0.1% and 0.05% magnesium and calcium chloride respectively at 163

20°C for 10 minutes before transferring to an ice bath for the remainder of 2 hours. The 164

spores were pelleted again at 3000rpm and rinsed three times for 10 minutes each in 165

sodium cacodylate buffer with added chlorides on ice. Secondary fixation with 1% 166

osmium tetroxide in sodium cacodylate buffer only was carried out at room temperature 167

for 1 hour. All following steps were performed at room temperature. Spores were rinsed 3 168

times in cacodylate buffer over 30 minutes and mordanted with 1% tannic acid for 30 169

minutes followed by a rinse with 1% sodium sulfate for 10 minutes. The samples were 170

dehydrated through an ethanol series 20%, 30% (staining en bloc with 2% uranyl acetate 171

at this stage), 50%, 70%, 90% and 95% for 20 minutes each then 100% for 3 x 20 172

minutes. This step and all future steps were carried out on a rotator to aid infiltration 173

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through the spore coat. Ethanol was exchanged for propylene oxide (PO) for 2 x 15 174

minutes followed by 1:1 PO to Epon resin mixture for at least 1 hour and neat Epon (with 175

a few drops of PO) over night. The spores were embedded in a flat moulded tray with 176

fresh resin and cured in an oven at 65°C for 24 hours. 40 nm sections were cut on a Leica 177

UCT ultramicrotome, contrasted with uranyl acetate and lead citrate and imaged on an 178

FEI 120 kV Spirit Biotwin using an F415 Tietz CCD camera. 179

180

Infective dose determination for environmental spore contamination 181

Spores from purified stock or feces of mice shedding high levels of C. difficile 182

630 were suspended in 10 ml of sterile PBS at levels of ~5, 50, 500, 5 000, 50 000 and 183

500 000 spores per 10 ml. To enumerate spores from feces, fecal pellets were suspended 184

in PBS to 100mg/ml. These samples were mixed 1:1 with 100% ethanol for 1 hour to kill 185

the vegetative cells. Samples were then washed in sterile PBS and then serially diluted 186

and plated as described for pure spore preps. 187

Within a sterile biosafety cabinet, each spore suspension was poured into a sterile 188

polysulfone cage (without bedding) and spread evenly to cover the entire bottom surface 189

(800 cm2). Contaminated cages were left to dry overnight resulting in a level of 190

environmental spore contamination of 0.006, 0.06, 06, 6, 60 and 600 spores per cm2. 191

Naïve mice (n=3) were aseptically (i.e. in biosafety cabinet while wearing a clean smock 192

and gloves that were disinfected with 2% Virkon) placed into each cage and left for 1 193

hour. Mice were then aseptically removed and individually placed in sterile cages with 194

standard bedding and food, and water that contained 250 mg/ml of clindamycin. C. 195

difficile infected mice generally shed low-levels of bacteria (~500 CFU/gram of feces). 196

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However, clindamycin treatment suppresses the intestinal microbiota and allows the 197

clindamycin resistant C. difficile 630 to overgrow within the murine intestinal tract 198

(Lawley et al. submitted). After 4 days of clindamycin treatment the feces of mice was 199

cultured for C. difficile on the selective Brazier’s agar plates. Mice that acquired C. 200

difficile 630 from the contaminated cage would be shedding >108 CFU C. difficile per 201

gram of feces whereas mice not colonized would not be shedding C. difficile (detection 202

limit of <101 CFU C. difficile per gram of feces). A fuller description of this murine 203

infection model is described elsewhere (Lawley et al., submitted for publication to 204

Infection and Immunity). All animal infections were performed in accordance with the 205

UK Home Office Animals (Scientific Procedures) Act of 1986. 206

207

Spore protein solubilisation and analysis 208

Purified spore pellets were re-suspended with NuPAGE Lithium dodecyl sulphate 209

(LDS) sample buffer (Invitrogen) containing 10% 2-mercaptoethanol and heated at 90˚C 210

for 10 minutes. The sample was centrifuged at 14 000 rpm for 15 minutes; the 211

supernatant is referred to as the LDS “soluble fraction” (Figure 4 b) and pellet 212

corresponds to insoluble fraction. The insoluble “pellet” (Figure 4 b) was further treated 213

with 8M urea /1M Na2CO3 and incubated for 10 minutes at room temperature and 214

centrifuged as before. The supernatant is referred to as “urea soluble fraction” (Figure 4). 215

The “urea insoluble pellet” (Figure 4) was saved for further processing as described 216

below. 217

The two soluble fractions were separated in a 4-12% NuPAGE Bis-Tris gel 218

(Invitrogen). Gels were stained overnight with colloidal Coomassie blue (Sigma). Each 219

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lane was excised into 20 bands that were in-gel digested overnight with trypsin 220

(sequencing grade; Roche). Peptides were extracted from gel bands twice with 50% 221

acetonitrile/0.5% formic acid and dried in a SpeedVac . 222

The “urea insoluble pellet” (from above) was washed three times with HPLC 223

water to remove detergent and then treated with 60% formic acid for 1 hour at 25˚C and 224

the suspension was neutralized with ammonium hydroxide solution (Fluka). Digestion 225

was performed overnight with trypsin (modified sequence grade, Promega). The sample 226

was centrifuged to remove any insoluble components and the supernatant, termed “tryptic 227

digest” (Figure 4 b). 228

229

Mass Spectrometry 230

The extracted peptides were re-dissolved in 0.5% formic acid (FA) and analyzed 231

with on-line nanoLC-MS/MS on an Ultimate 3000 Nano/Capillary LC System (Dionex) 232

coupled to a LTQ FT Ultra mass spectrometer (ThermoElectron) that is equipped with a 233

nanospray ion source. Samples were separated on a RP analytical column (BEHC18 75 234

µm id x 10 cm) (Waters) over a 45 min (for weak gel bands) or 60 min (for intense gel 235

bands) at a linear gradient of 4-32% CH3CN/0.1% FA, or for 120 minutes for the pellet 236

digest. 237

The mass spectrometer was operated in the standard data dependent acquisition 238

mode controlled by Xcalibur 2.0. The instrument was operated with a cycle of one MS (in 239

the FTICR cell) acquired at a resolution of 100,000 at m/z 400. The top three (for gel 240

band extracts) or top five (for pellet digest) most abundant multiply-charged ions were 241

subject to MS/MS in the linear ion trap. 242

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The raw data files were processed by BioWorks 3.3 (ThermoElectron) and then 243

submitted to a database search in Mascot server 2.2 (www.matrixscience.com) against an 244

in-house built C. difficile database using following search parameters: trypsin/P with 3 245

mis-cleavage, 20 ppm for MS, 0.5 Da for MS/MS, with 9 variable modification of Acetyl 246

(N-term), Carbamidomethyl (C), Deamidated (NQ), Dioxidation (M), Formyl (N-term), 247

Gln->pyro-Glu (N-term Q), Glu->pyro-Glu (N-term E), Methyl (E) and Oxidation (M). 248

Protein identifications were filtered using 5% FDR per peptide from reverse database 249

search in Mascot and combined with manual checking for single peptide matches based 250

protein identifications. 251

252

Bioinformatic analysis 253

Analysis of whole genome and spore proteome content was performed using Artemis (8). 254

Repicrocal FASTA analysis was performed as previously described (33) using the 255

genomes of C. acetobutylicum (AE001437), C. bartlettii (B0A9S8), C. beijerinckii 256

(CP000721), C. botulinum (AM412317), C. cellulolyticum (CP001348), C. kluyveri 257

(CP000673), C. novyi (CP000382), C. perfringens (CP000246), C. phytofermentans 258

(CP000885), C. tetani (AE015927), C. thermocellum (CP000568) and B. subtilis 259

(AL09126). 260

261

RESULTS 262

Purification of spores from C. difficile 630 263

Since little is known about the structure and composition of C. difficile spores we 264

developed a protocol for their purification. C. difficile 630 was selected for the initial 265

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analysis as the complete genome sequence is available (34). Routine culture experiments 266

using C. difficile 630 grown statically for 5-7 days in Wilson’s broth (45) showed that the 267

strain reproducibly produced ~106-107 spores/ml, which was 10-100-fold higher 268

compared to the same strain grown in BHI broth (data not shown). We therefore grew C. 269

difficile 630 for 7 days in Wilson’s broth prior to spore purification. 270

Initial attempts to purify C. difficile spores were guided by an established B. 271

subtilis spore purification protocol (9). This protocol exploits serial washing of bacterial 272

cells in cold water to lyse vegetative cells, followed by density gradient centrifugation to 273

separate spores from the remaining vegetative cells and debris. Although we found that 274

this protocol did enrich for C. difficile spores there was significant contamination with 275

dead vegetative cells and an unidentified extracellular matrix that appeared to facilitate 276

the clumping of bacteria (Figure 1a). Therefore, to isolate highly purified spores, we 277

developed a protocol based on the sequential treatment of C. difficile cultures with 278

detergent, lysozyme, sonication and proteinase K followed by sucrose layer 279

centrifugation (see Materials and Methods). This protocol effectively and reproducibly 280

yielded spores of a high purity (>99.99% free from vegetative cells; Figure 1a and b). 281

Transmission electron microscopic examination of spores showed they were 282

composed of an electron dense outer surface with a spore coat structure composed of 283

concentric inner rings (Figure 1c and d). The outer surface of the spore coat often 284

contained an exosporium (Figure 1c). Within the spore coat was a clearer cortex region 285

demarked by a putative inner membrane that defines the core. Within the core were 286

globular-like structures that appeared to be densely packed ribosomes, with intervening 287

regions that likely contain condensed nucleoprotein complexes. A thorough examination 288

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of the microscope grids failed to identify any evidence of vegetative cells or obvious cell 289

debris. Thus, using the novel protocol we were able to isolate intact and well-defined C. 290

difficile spores at a high level of purity. 291

292

Pure C. difficile spores are viable and maintain their resistant nature 293

The number of spores yielded within individual preparations was routinely 294

estimated by counting samples visually using a light microscope, as spores are easily 295

recognized by their characteristic phase bright appearance (23). Next we sought to 296

determine if the spores remained viable after exposure to the relatively harsh purification 297

conditions. Compared to the number of spores estimated by visual counting, only ~0.1-298

1% of the spores routinely germinated and grew out as colonies on BHI agar plates 299

(Figure 2a). However, by supplementing BHI agar with 0.1% of the germinants cholate, 300

taurocholate or glycocholate (40), we observed a 100 to 1,000-fold increase in 301

germination and outgrowth efficiency. Hence, the purified spores remain viable and 302

retain their sensitivity to cholate-based germinants (40, 45). 303

The ability to prepare pure spores allowed us to quantitatively evaluate the 304

sensitivity of spores to different chemical or physical treatments. Heating cultures of 305

Clostridium or Bacillus spp. to 60°C for 10 minutes is a routine method of inactivating 306

vegetative cells while retaining the spores in a viable state (23, 40). To test if purified C. 307

difficile spores remain resistant to high temperatures, they were incubated in water heated 308

to 60°C or 70°C. Significantly, C. difficile 630 spores remained completely viable when 309

incubated at 60°C for up to 24 hours (Figure 2b). Even at 70°C, high levels of C. difficile 310

were recovered after 3 hours of incubation but these levels dropped to <1% of the input 311

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after 24 hours (Figure 2b). Further, we found that 70% ethanol had no obvious or 312

reproducible effect on the viability of the spores whereas the sporicidal agent Virkon 313

(1%) efficiently inactivated the spores (Figure 2c). Therefore, the purified spores are 314

resistant to prolonged exposure to high temperatures and 70% ethanol but are effectively 315

inactivated by sporicidal agents. 316

317

Environmental C. difficile spores are highly infective 318

We next evaluated the host infectivity of purified spores by calculating the dose 319

required to infect laboratory mice. To do so, we contaminated individual cages, without 320

bedding, with differing concentrations of spores. Naïve recipient mice were aseptically 321

placed in contaminated cages for 1 hour and then these mice were placed individually in 322

sterile cages for 4 days and provided with water containing clindamycin. C. difficile 630 323

is resistant to clindamycin so treatment of mice that acquired spores from the 324

contaminated cages triggers C. difficile overgrowth and high level fecal shedding (>108 325

CFU C. difficile/gram feces)(Lawley et al., submitted for publication to Infection and 326

Immunity). In contrast, mice that did not acquire spores from contaminated cages would 327

not shed any C. difficile after clindamycin treatment. 328

Based on these experiments we found that environmental spores infected mice in 329

a dose-dependant manner (Figure 3a). Using the Reed-Muech formulation (25) we 330

determined the dose required to infect 50% of the mice (ID50) under these conditions is 331

~7 spores/cm2 (Figure 3a). This ID50 is comparable to that determined from C. difficile 332

630 spores derived from the feces of C. difficile infected mice (4 spores/cm2)(Figure 3a). 333

Importantly, infection of mice with pure spores resulted in asymptomatic intestinal 334

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carriage of C. difficile (~500 CFU C. difficile/gram feces) that lasted several weeks after 335

cessation of clindamycin treatment (Figure 3b). These results demonstrate that purified 336

spores are highly infective to mice. 337

338

Determination of the C. difficile spore proteome 339

The proteome profiles resolved by SDS-PAGE of vegetative cells and purified 340

spore preparations are shown in Figure 4a. The proteome from vegetative cells shows a 341

very dense banding pattern due to a more complex protein composition when compared 342

to that from pure spores. Moreover, prominent polypeptide signatures do not overlap 343

between the two samples indicating the profiles are distinct and therefore are of very 344

different composition. To further define the structure and composition of purified C. 345

difficile spores, an exhaustive proteomic analysis was performed by tandem mass 346

spectrometry (LC-MS/MS) as outlined in Figure 4b. Spore proteins were extracted using 347

two solublization steps, first exploiting standard NuPAGE sample buffer LDS (with 10% 348

2-mercaptoethanol) and subsequently using 8M urea in 1M Na2CO3. Polypeptides in 349

these fractions were separated using SDS-PAGE and analysed by LC-MS/MS. The first 350

solubilization step produced ~233 polypeptides and the second step an additional 71 351

polypeptides (Figure 4b). Direct tryptic digestion was performed on the remaining 352

insoluble pellet and analysis of this sample lead to further assignment of 32 polypeptides. 353

Multiple treatment steps were used to ensure thorough analysis of the spore proteome 354

(Figure 4c). There are no obvious trends between spore protein solubility and 355

functionality (see color coding in Supplemental Table 1). Overall, ~336 proteins were 356

identified as components of purified C. difficile 630 spores. 357

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358

Genome mapping of the C. difficile spore proteome 359

In order to analyse the spore proteome further, the spore-associated polypeptides 360

were mapped onto the annotated C. difficile 630 genome (Figure 5)(34). The genes 361

encoding the spore-associated polypeptides are dispersed throughout the C. difficile 630 362

genome with a notable cluster of ribosomal genes near the origin of replication (Figure 363

5a). The proteomics data revealed three novel small proteins that were not previously 364

identified in the annotated genome (34). The CDS encoding these proteins were named 365

CD1595A, CD2687A and CD3439A. 366

Classifying the spore-associated polypeptides according to the functional scheme 367

used in the annotated genome revealed a number of trends (Figure 6 and supplemental 368

Table 1). Of the 60 predicted proteins in the sporulation/germination class that are present 369

in the entire genome (34), fourteen (~25%) were detected as spore-associated peptides 370

(Figure 6 and supplemental Table 1). Many of the in silico predicted sporulation proteins 371

apparently absent from the purified spores are likely associated with the earlier stages of 372

spore formation in other bacteria (42), hence these would not necessarily be expected to 373

be present in the mature C. difficile spore structure. 374

The most common functional class of the spore proteome, comprising 25% of all 375

the detected polypeptides, were those involved in translation (Figure 6 and supplemental 376

Table 1). This is in agreement with the electron microscopic analysis that revealed a high 377

density of ribosomes in the core cytoplasm of the C. difficile spores. Interestingly, the 378

majority (15 out of 16) of the total predicted chaperone complement of C. difficile was 379

found to be present in the spore proteome (Figure 6), suggesting the importance of 380

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maintaining the folded structure of proteins in these dormant bodies during harsh 381

conditions and within the densely packed spore core. Proteases were also relatively 382

abundant, with 16 of 40 annotated in the genome present (Figure 6) (>2.5 fold over-383

representation). Stress response proteins including ruberythrins, tellurium resistance 384

proteins, bacterioferritin, catalase and superoxide dismutase are present within the spore 385

and may contribute to the protection of spores from oxidative stress during germination 386

(supplemental Table 1). 387

Proteins involved in regulation are under-represented in the spore proteome 388

(Figure 6). The late sporulation-specific sigma factor σG (SigG) was the only putative 389

sigma factor detected and peptides for only two predicted transcription factors were 390

identified. These were CcpA (Figure 5b), a homologue of the carbon catabolite repressor 391

important for sporulation in C. perfringens (43) and CodY, the GTP- and branched chain 392

amino acid-activated pleiotropic transcriptional repressor of early stationary phase genes 393

(10). The presence of CodY in C. difficile spores suggests that during germination the 394

spore represses genes associated with sporulation and stationary phase growth. 395

A total of 41 hypothetical proteins were detected in the proteome (Figure 6), 396

validating the annotation of these coding sequences (34). One of the more abundant spore 397

proteins was the hypothetical protein CD1067, a 405 aa cysteine-rich protein (10% cys) 398

that is encoded within a distinct genomic region containing several spore genes (Figure 399

5b). Despite being present in the spore preparations at high levels, coverage across the 400

length of the CD1067 protein was distinctly non-uniform, with peptides rich in cysteine 401

residues infrequently detected, regardless of the presence of 2-mercaptoethanol in the 402

solubilization buffer. This suggests these sulphur-containing residues may not be 403

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accessible to trypsin digestion and might be involved in extensive disulphide bridge 404

formation, or are otherwise modified in the spore. CD1067 has an orthologue in C. 405

bartlettii but no other sequenced bacterium, including Clostridium or Bacillus spp. 406

Interestingly, the N-terminal region of the protein encoded by CD1067 also shows limited 407

similarity (52%) to a small cysteine-rich outer membrane protein OmcA from Chlamydia 408

trachomatis that provides structural integrity to the outer envelope of the infectious 409

elementary body (1). 410

Metabolic proteins are well represented with 34 being classified in energy and 20 411

in central/intermediary metabolism (Figure 6 and supplemental Table 1). Anabolic 412

pathways, such as purine, pyrimidine and amino acid biosynthesis, are poorly 413

represented, whilst many of the components of the catabolic glycolytic pathway were 414

detected. Also present are enzymes required for the fermentation of amino acids via the 415

Stickland reactions (16, 17, 19), including CoA dehydrogenase enzymes and electron 416

transfer proteins (CD1054-1059) (18)(Figure 5b). KEGG pathway analysis 417

(http://www.genome.ad.jp/kegg)(supplemental Table 1) demonstrated that nearly 418

complete pathways for gluconeogenesis (production of glucose from pyruvate) and 419

carbon fixation as well as enzymes that convert D-lactate, phosphoenolpyruvate and 420

formate into pyruvate are present within spores. Further, the nearly complete pentose 421

phosphate pathway (production of ribose and NADPH from glucose) is also present 422

(supplemental Table 1). 423

In total, 39 of the spore-associated proteins are predicted to be secreted or surface-424

associated (Figure 6). In accordance with this observation, SecA, involved in the 425

translocation of proteins across the cell membrane, and a foldase (PrsA) were also 426

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represented in the dataset. Almost half of these (21 CDSs) have no predicted function in 427

the genome annotation. Of those that do, six are predicted substrate-binding proteins for 428

ABC transporters. Only one transporter (CD2465), predicted to facilitate the import of 429

amino acids, is found in the spore proteome. Interestingly, a subset of the surface 430

associated proteins contain functional domains that are generally associated with 431

adherence to host cells, such the collagen-like glycoprotein BclA (CD0332), two surface 432

layer proteins (CD2791 and CD2793 (SlpA)) that contain CW binding domains and a 433

putative hemagglutanin/adhesin (CD0514) (supplemental Table 1). 434

435

Comparison of C. difficile spore genes to other clostridial genomes 436

We performed reciprocal FASTA analysis to compare the C. difficile genes that 437

encode the spore proteome to the sequenced genomes of 11 clostridial species including 438

C. acetobutylicum, C. bartlettii, C. beijerinckii, C. botulinum, C. cellulolyticum, C. 439

kluyveri, C. novyi, C. perfringens, C. phytofermentans, C. tetani and C. thermocellum. 440

We found that 51-70% of the C. difficile spore proteome genes have orthologues in 441

individual clostridia (supplemental Figure 1). However, only 26% (88 of 336) of the C. 442

difficile spore proteome genes are well conserved among all the tested clostridia genomes 443

(supplemental Table 1). The majority of the C. difficile spore genes that are conserved in 444

the 11 clostridial genomes encode proteins associated with translation, protein folding 445

and degradation and glycolysis (Figure 7a). The C. bartletti genome contains the highest 446

proportion of orthologues (70%) shared with the C. difficile spore proteome 447

(supplemental Figure 1). C. bartletti is a component of the human intestinal microbiota 448

and is phylogenetically the closest available clostridial genome to C. difficile (16S rDNA 449

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place these two species in cluster XI (38)). Interestingly, among the 336 genes of the C. 450

difficile spore proteins, 17 (5% of C. difficile spore proteome) have orthologues only in C. 451

bartletti (Figure 7b). The majority of these genes encode proteins with no known 452

function, including the C. difficile gene CD1067 encoding the cysteine-rich protein 453

discussed above. However, many of the genes encode proteins associated with 454

macromolecular degradation, such as putative chitinase/thioredoxin (CD1433), 455

polysaccarhide deacetylase (1319), peptidases (CD0684, CD3221, CD2246) and an 456

amidohydrolase (CD2841) (supplemental Table 1). 457

We found that 29 spore genes, representing 9% of the spore proteome, are 458

currently unique to C. difficile relative to the representative clostridial genomes (Figure 459

7c and supplemental Table 1). Many of these genes encode hypothetical proteins. 460

Interestingly, 5 genes unique to C. difficile are encoded within various mobile elements, 461

including those from Prophage 1 (CD0939 and CD0940), a genomic island (CD1898; 462

putative phage endolysin), Tn5398 (CD2010; erythromycin resistance factor; note that 463

CD2010 and CD2007 are identical but the latter is likely not expressed due to a potential 464

promoter deletion (12)) and prophage 2 (CD2926) (supplemental Table 1), although no 465

spore-associated genes mapped to any of the seven conjugative transposons (Figure 5). 466

These observations imply that the acquisition of some classes of mobile elements by C. 467

difficile can directly contribute to the mature spore composition. Importantly, predicted 468

surface exposed proteins represent 17% (5 of 29) of the unique C. difficile spore 469

proteome, including a putative exosporium glycoprotein BclA1 (CD0332), the S-layer 470

protein SlpA (CD2793) (11) and its paralogue CD2791. 471

472

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DISCUSSION 473

Here we describe a novel protocol for the isolation of highly purified spores from 474

cultures of a human virulent C. difficile. Purified spores remain viable for extended 475

periods of time, respond to cholate germinants and retain their resistances to heat and 476

ethanol. The availability of purified spores facilitated for the first time an estimate of the 477

infectious dose of this pathogen, as assessed by an assay that mimics environmental spore 478

contamination. Hence, the C. difficile spores were not irreversibly damaged or 479

significantly altered by the purification process. This spore purification protocol will be 480

valuable to purify spores from other C. difficile strains, particularly those with genomes 481

being sequenced (http://www.sanger.ac.uk/Projects/C_difficile), as well as spores from 482

other clostridial species. The availability of pure C. difficile spores also facilitated the 483

first detailed proteomic and bioinformatic analysis of spores from any Clostridia. The 484

spore proteome of ~336 polypeptides defined here likely harbour proteins that contribute 485

to the high level of resistance displayed by spores during host-to-host transmission and 486

survival in the hospital environment, as well as those that stimulate germination and 487

tissue colonization after entry into the mammalian intestinal tract. 488

C. difficile does not encode the classical germination receptors present in Bacilli 489

and many Clostridia species (33, 34). Perhaps this implies a novel mechanism of 490

germination by C. difficile spores. C. difficile spores germinate in response to bile-491

associated cholate derivatives in vitro (Figure 2) (40) suggesting that C. difficile spores 492

germinate within the intestinal tract. Thus, C. difficile spores are likely to possess a novel 493

receptor(s) for cholate-based compounds. There are 29 predicted cell surface proteins and 494

12 predicted transport/binding proteins within the spore-associated proteome that could 495

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directly engage with the extracellular environment and are therefore considered candidate 496

germination receptors. The abundance of ribosome-associated proteins, translation factors 497

and RNA polymerases indicates that high levels of translation are required to drive 498

primary biological processes upon germination. The presence of apparently intact 499

ribosomes within the spore indicates that these molecular machines are robust enough to 500

survive the purification process that destroys the vegetative cells. The profusion of 501

chaperones in the C. difficile spore proteome probably plays a crucial role in preventing 502

the unfolding of proteins during germination. 503

Analysis of the spore proteome suggests that amino acid fermentation (Stickland 504

reaction), to produce energy, and pyruvate metabolism, to produce carbohydrates, are 505

important early requirements for a germinating C. difficile spore. To supply the 506

translation machinery and Stickland reactions (16, 17) with substrates spores may rely on 507

stored reserves of amino acids present within intact proteins. This hypothesis is supported 508

by the presence of proteases/peptidases within the spore proteome that could degrade 509

spore proteins during the regeneration of vegetative cells. Indeed, Bacilli and Clostridia 510

spores are known to degrade specific proteins during germination (31, 32, 37). 511

Surprisingly, there are very few proteins within the spore that are dedicated to DNA 512

replication, cell division and transcriptional regulation (Figure 5). The lack of proteins 513

involved in transcriptional regulation suggests that during germination the initial 514

transcriptional patterns are limited and likely pre-determined. Therefore, upon 515

germination C. difficile spores likely undergo a period of macromolecular degradation, 516

amino acid fermentation, pyruvate metabolism and translation prior to engaging in 517

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transcription, replication and cell division. Interestingly, DNA replication initiates after a 518

lag period upon germination of B. megaterium spores (35). 519

One of the more abundant spore proteins was the cysteine-rich hypothetical 520

protein CD1067. C. bartlettii is the only other bacteria that possesses an orthologue (42% 521

identity) although we did note limited homology to the surface protein OmcA from the 522

transmissible form of C. trachomatis (1). Interestingly, B. subtilis spores contain the 523

cysteine-rich proteins CotY and CotZ (these have no detectable homology with CD1067) 524

in their outer spore coat that influence the accessibility of germinants to their receptors 525

(46). Thus, it may be envisaged that a highly abundant and cysteine-rich surface protein 526

could form a rigid, disulphide-linked mesh structure around the C. difficile spore in 527

oxidative conditions (i.e. environment) but become weakened in reduced conditions (i.e. 528

intestinal tract) to facilitate germination. If so, CD1067 is a candidate marker for C. 529

difficile spore detection. Further, unique surface exposed proteins on spores are also 530

excellent candidates for reverse vaccinology (28) to develop a protective vaccine to C. 531

difficile. A prime antigen candidate for the C. difficile spore is the collagen-like protein 532

BclA1 that has an orthologue in B. anthracis spores. Immunization with BclA protects 533

animals from B. anthracis spore colonization by increasing opsonophagocytic uptake of 534

spores and inhibiting intramacrophage germination (6, 14). Another candidate is the 535

unique C. difficile surface layer protein SlpA since it is present on the surface of 536

vegetative cells (11) and also in spores of C. difficile. 537

Our previous bioinformatic analysis of the genome of C. difficile 630 has shown 538

that many of the genes encoding classical spore proteins found in other bacteria are not 539

obviously present in the C. difficile 630 genome (33, 34). For example, orthologues of 540

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only 18 of 70 B. subtilis spore coat proteins could be identified in the genome of C. 541

difficile 630 (15). Our comparative genome analysis identified only 88 C. difficile spore 542

genes with orthologues present in the genomes of 11 Clostridium species. These genes 543

should represent the core spore genes that were inherited from the common ancestor of 544

the clostridia. The majority of these genes encode proteins that function in the production, 545

stability and degradation of spore proteins, likely reflecting the importance of protein 546

turnover during clostridial sporulation and germination. 547

The C. difficile 630 spore proteome contains several unique features that could be 548

exploited for the detection and discrimination of C. difficile spores, providing candidate 549

targets for future diagnostic tools. Current diagnostics are based on antibody detection of 550

C. difficile toxins A and B in patient stool, generally to confirm C. difficile as the 551

causative agent of diarrhea (26). Accurate detection of C. difficile spores from hospital 552

patients, especially those excreting high-levels spores, and from the environmental should 553

improve infection control measures targeted at preventing C. difficile disease in hospitals. 554

555

ACKNOWLEDGEMENTS 556

We are grateful to Washington University Genome Sequencing Center for permission to 557

use the C. bartlettii genome data. We thank Neil Fairweather and Stephen Bentley for 558

comments on the manuscript and Cordellia Brandt, Nicola Goodwin and Claire Raisen 559

for assistance with the animal experiments. This work was funded by the Wellcome Trust 560

and a Royal Society of London fellowship to TDL. 561

562

REFERENCES 563

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1. Allen, J. E., M. C. Cerrone, P. R. Beatty, and R. S. Stephens. 1990. Cysteine-564 rich outer membrane proteins of Chlamydia trachomatis display compensatory 565 sequence changes between biovariants. Mol Microbiol 4:1543-50. 566

2. Bartlett, J. G. 2006. Narrative review: the new epidemic of Clostridium difficile-567 associated enteric disease. Ann Intern Med 145:758-64. 568

3. Bartlett, J. G., T. Chang, N. S. Taylor, and A. B. Onderdonk. 1979. Colitis 569 induced by Clostridium difficile. Rev Infect Dis 1:370-8. 570

4. Borriello, S. P. 1998. Pathogenesis of Clostridium difficile infection. J 571 Antimicrob Chemother 41 Suppl C:13-9. 572

5. Borriello, S. P., and P. Honour. 1981. Simplified procedure for the routine 573 isolation of Clostridium difficile from faeces. J Clin Pathol 34:1124-7. 574

6. Brahmbhatt, T. N., S. C. Darnell, H. M. Carvalho, P. Sanz, T. J. Kang, R. L. 575 Bull, S. B. Rasmussen, A. S. Cross, and A. D. O'Brien. 2007. Recombinant 576 exosporium protein BclA of Bacillus anthracis is effective as a booster for mice 577 primed with suboptimal amounts of protective antigen. Infect Immun 75:5240-7. 578

7. Brazier, J. S. 2008. Clostridium difficile: from obscurity to superbug. Br J 579 Biomed Sci 65:39-44. 580

8. Carver, T., M. Berriman, A. Tivey, C. Patel, U. Bohme, B. G. Barrell, J. 581 Parkhill, and M. A. Rajandream. 2008. Artemis and ACT: viewing, annotating 582 and comparing sequences stored in a relational database. Bioinformatics 24:2672-583 6. 584

9. Coleman, W. H., D. Chen, Y. Q. Li, A. E. Cowan, and P. Setlow. 2007. How 585 moist heat kills spores of Bacillus subtilis. J Bacteriol 189:8458-66. 586

10. Dineen, S. S., A. C. Villapakkam, J. T. Nordman, and A. L. Sonenshein. 2007. 587 Repression of Clostridium difficile toxin gene expression by CodY. Mol 588 Microbiol 66:206-19. 589

11. Fagan, R. P., D. Albesa-Jove, O. Qazi, D. I. Svergun, K. A. Brown, and N. F. 590 Fairweather. 2009. Structural insights into the molecular organization of the S-591 layer from Clostridium difficile. Mol Microbiol 71:1308-22. 592

12. Farrow, K. A., D. Lyras, and J. I. Rood. 2000. The macrolide-lincosamide-593 streptogramin B resistance determinant from Clostridium difficile 630 contains 594 two erm(B) genes. Antimicrob Agents Chemother 44:411-3. 595

13. Gerding, D. N., C. A. Muto, and R. C. Owens, Jr. 2008. Measures to control 596 and prevent Clostridium difficile infection. Clin Infect Dis 46 Suppl 1:S43-9. 597

14. Hahn, U. K., R. Boehm, and W. Beyer. 2006. DNA vaccination against anthrax 598 in mice-combination of anti-spore and anti-toxin components. Vaccine 24:4569-599 71. 600

15. Henriques, A. O., and C. P. Moran, Jr. 2007. Structure, assembly, and function 601 of the spore surface layers. Annu Rev Microbiol 61:555-88. 602

16. Jackson, S., M. Calos, A. Myers, and W. T. Self. 2006. Analysis of proline 603 reduction in the nosocomial pathogen Clostridium difficile. J Bacteriol 188:8487-604 95. 605

17. Jackson-Rosario, S., D. Cowart, A. Myers, R. Tarrien, R. L. Levine, R. A. 606 Scott, and W. T. Self. 2009. Auranofin disrupts selenium metabolism in 607 Clostridium difficile by forming a stable Au-Se adduct. J Biol Inorg Chem 608 14:507-19. 609

on October 24, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

18. Karlsson, S., L. G. Burman, and T. Akerlund. 2008. Induction of toxins in 610 Clostridium difficile is associated with dramatic changes of its metabolism. 611 Microbiology 154:3430-6. 612

19. Kim, J., D. J. Darley, W. Buckel, and A. J. Pierik. 2008. An allylic ketyl 613 radical intermediate in clostridial amino-acid fermentation. Nature 452:239-42. 614

20. Li, J., and B. A. McClane. 2008. A novel small acid soluble protein variant is 615 important for spore resistance of most Clostridium perfringens food poisoning 616 isolates. PLoS Pathog 4:e1000056. 617

21. Loo, V. G., L. Poirier, M. A. Miller, M. Oughton, M. D. Libman, S. Michaud, 618

A. M. Bourgault, T. Nguyen, C. Frenette, M. Kelly, A. Vibien, P. Brassard, S. 619 Fenn, K. Dewar, T. J. Hudson, R. Horn, P. Rene, Y. Monczak, and A. Dascal. 620 2005. A predominantly clonal multi-institutional outbreak of Clostridium difficile-621 associated diarrhea with high morbidity and mortality. N Engl J Med 353:2442-9. 622

22. McDonald, L. C., G. E. Killgore, A. Thompson, R. C. Owens, Jr., S. V. 623 Kazakova, S. P. Sambol, S. Johnson, and D. N. Gerding. 2005. An epidemic, 624 toxin gene-variant strain of Clostridium difficile. N Engl J Med 353:2433-41. 625

23. Moir, A. 1981. Germination properties of a spore coat-defective mutant of 626 Bacillus subtilis. J Bacteriol 146:1106-16. 627

24. Moir, A. 2006. How do spores germinate? J Appl Microbiol 101:526-30. 628 25. Ozanne, G. 1984. Estimation of endpoints in biological systems. Comput Biol 629

Med 14:377-84. 630 26. Planche, T., A. Aghaizu, R. Holliman, P. Riley, J. Poloniecki, A. Breathnach, 631

and S. Krishna. 2008. Diagnosis of Clostridium difficile infection by toxin 632 detection kits: a systematic review. Lancet Infect Dis 8:777-84. 633

27. Plomp, M., T. J. Leighton, K. E. Wheeler, H. D. Hill, and A. J. Malkin. 2007. 634 In vitro high-resolution structural dynamics of single germinating bacterial spores. 635 Proc Natl Acad Sci U S A 104:9644-9. 636

28. Rappuoli, R. 2000. Reverse vaccinology. Curr Opin Microbiol 3:445-50. 637 29. Riggs, M. M., A. K. Sethi, T. F. Zabarsky, E. C. Eckstein, R. L. Jump, and C. 638

J. Donskey. 2007. Asymptomatic carriers are a potential source for transmission 639 of epidemic and nonepidemic Clostridium difficile strains among long-term care 640 facility residents. Clin Infect Dis 45:992-8. 641

30. Rupnik, M. 2007. Is Clostridium difficile-associated infection a potentially 642 zoonotic and foodborne disease? Clin Microbiol Infect 13:457-9. 643

31. Sanchez-Salas, J. L., M. L. Santiago-Lara, B. Setlow, M. D. Sussman, and P. 644 Setlow. 1992. Properties of Bacillus megaterium and Bacillus subtilis mutants 645 which lack the protease that degrades small, acid-soluble proteins during spore 646 germination. J Bacteriol 174:807-14. 647

32. Sanchez-Salas, J. L., and P. Setlow. 1993. Proteolytic processing of the protease 648 which initiates degradation of small, acid-soluble proteins during germination of 649 Bacillus subtilis spores. J Bacteriol 175:2568-77. 650

33. Sebaihia, M., M. W. Peck, N. P. Minton, N. R. Thomson, M. T. Holden, W. J. 651

Mitchell, A. T. Carter, S. D. Bentley, D. R. Mason, L. Crossman, C. J. Paul, 652 A. Ivens, M. H. Wells-Bennik, I. J. Davis, A. M. Cerdeno-Tarraga, C. 653 Churcher, M. A. Quail, T. Chillingworth, T. Feltwell, A. Fraser, I. 654 Goodhead, Z. Hance, K. Jagels, N. Larke, M. Maddison, S. Moule, K. 655

on October 24, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

Mungall, H. Norbertczak, E. Rabbinowitsch, M. Sanders, M. Simmonds, B. 656 White, S. Whithead, and J. Parkhill. 2007. Genome sequence of a proteolytic 657 (Group I) Clostridium botulinum strain Hall A and comparative analysis of the 658 clostridial genomes. Genome Res 17:1082-92. 659

34. Sebaihia, M., B. W. Wren, P. Mullany, N. F. Fairweather, N. Minton, R. 660

Stabler, N. R. Thomson, A. P. Roberts, A. M. Cerdeno-Tarraga, H. Wang, 661 M. T. Holden, A. Wright, C. Churcher, M. A. Quail, S. Baker, N. Bason, K. 662 Brooks, T. Chillingworth, A. Cronin, P. Davis, L. Dowd, A. Fraser, T. 663 Feltwell, Z. Hance, S. Holroyd, K. Jagels, S. Moule, K. Mungall, C. Price, E. 664 Rabbinowitsch, S. Sharp, M. Simmonds, K. Stevens, L. Unwin, S. Whithead, 665 B. Dupuy, G. Dougan, B. Barrell, and J. Parkhill. 2006. The multidrug-666 resistant human pathogen Clostridium difficile has a highly mobile, mosaic 667 genome. Nat Genet 38:779-86. 668

35. Setlow, P. 1973. Deoxyribonucleic acid synthesis and deoxynucleotide 669 metabolism during bacterial spore germination. J Bacteriol 114:1099-107. 670

36. Setlow, P. 2003. Spore germination. Curr Opin Microbiol 6:550-6. 671 37. Setlow, P., and W. M. Waites. 1976. Identification of several unique, low-672

molecular-weight basic proteins in dormant spores of Clostridium bifermentans 673 and their degradation during spore germination. J Bacteriol 127:1015-7. 674

38. Song, Y. L., C. X. Liu, M. McTeague, P. Summanen, and S. M. Finegold. 675 2004. Clostridium bartlettii sp. nov., isolated from human faeces. Anaerobe 676 10:179-84. 677

39. Songer, J. G., and M. A. Anderson. 2006. Clostridium difficile: an important 678 pathogen of food animals. Anaerobe 12:1-4. 679

40. Sorg, J. A., and A. L. Sonenshein. 2008. Bile salts and glycine as cogerminants 680 for Clostridium difficile spores. J Bacteriol 190:2505-12. 681

41. Sorg, J. A., and A. L. Sonenshein. 2009. Chenodeoxycholate is an inhibitor of 682 Clostridium difficile spore germination. J Bacteriol 191:1115-7. 683

42. Stragier, P., and R. Losick. 1996. Molecular genetics of sporulation in Bacillus 684 subtilis. Annu Rev Genet 30:297-41. 685

43. Varga, J., V. L. Stirewalt, and S. B. Melville. 2004. The CcpA protein is 686 necessary for efficient sporulation and enterotoxin gene (cpe) regulation in 687 Clostridium perfringens. J Bacteriol 186:5221-9. 688

44. Vonberg, R. P., E. J. Kuijper, M. H. Wilcox, F. Barbut, P. Tull, P. Gastmeier, 689

P. J. van den Broek, A. Colville, B. Coignard, T. Daha, S. Debast, B. I. 690 Duerden, S. van den Hof, T. van der Kooi, H. J. Maarleveld, E. Nagy, D. W. 691 Notermans, J. O'Driscoll, B. Patel, S. Stone, and C. Wiuff. 2008. Infection 692 control measures to limit the spread of Clostridium difficile. Clin Microbiol Infect 693 14 Suppl 5:2-20. 694

45. Wilson, K. H. 1983. Efficiency of various bile salt preparations for stimulation of 695 Clostridium difficile spore germination. J Clin Microbiol 18:1017-9. 696

46. Zhang, J., P. C. Fitz-James, and A. I. Aronson. 1993. Cloning and 697 characterization of a cluster of genes encoding polypeptides present in the 698 insoluble fraction of the spore coat of Bacillus subtilis. J Bacteriol 175:3757-66. 699

700 701

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Figure 1. Visualization of pure C. difficile spores. Endospore stain of a) C. difficile 702

culture illustrates the pink vegetative cells (veg) and pink extracellular matrix (ex) with 703

few interspersed green spores (sp). b) purified C. difficile spores are stained green. c) 704

TEM of sectioned C. difficile spores demonstrating the spore ultrastructure including the 705

exosporium, coat, cortex, core, membrane and ribosomes (scale bar = 100 nm). d) 706

magnified section of outer surface of spore (scale bar = 50 nm). 707

708

Figure 2 Pure C. difficile spores are viable and maintain resistant nature. a) 709

Germination of pure C. difficile spores in response to cholate, taurocholate and 710

glycocholate. Spores were serially diluted and plated on BHI agar plates containing 0.1% 711

of the indicated germinant. Spores were also enumerated visually with a light 712

microscope. b) heat resistance properties of spores. Spores were incubated in distilled 713

water heated to 60ºC or 70ºC for the indicated time. c) chemical resistance properties of 714

spores. Spores were incubated in water, 70% or 1% Virkon for 20 minutes at room 715

temperature. After incubation, spores were serially diluted and plated on BHI + 0.5% 716

taurocholate. Germinated spores were counted after 48 hours of growth at 37ºC under 717

anaerobic conditions. The detection limit of 5x101 C. difficile/ml is shown with a dashed 718

horizontal line. 719

720

Figure 3. Environmental C. difficile 630 spores are highly infective. a) Infective dose 721

curves of pure and fecal-derived C. difficile 630 spores in mice (n=3) after exposure to 722

environmental spore contamination. Based on the Reed-Muench Formulation the 723

environmental spore dose to infect 50% (ID50) of the mice within a 1-hour exposure is 4 724

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spores/cm2 for fecal spores and 7 spores/cm2 for pure spores, as indicated by the gray and 725

black vertical dashed lines. Data is representative of two independent experiments. b) 726

Fecal shedding of C. difficile 630 from infected mice (n=6) demonstrates that spores 727

establish intestinal carriage in infected mice. Broken gray line represents the expected 728

shedding pattern during the induction of the high level C. difficile shedding. The 729

detection limit of 5x101 C. difficile/gram feces is shown with a dashed horizontal line. 730

731

Figure 4. Summary of scheme to determine spore proteome. a) SDS-PAGE gel of 732

vegetative cells (left lane) and pure spores (right lane) after solubilization with LDS. b) 733

flow diagram outlining the steps required to solubilize proteins from C. difficile spores 734

and the number of extra proteins liberated at each stage. c) rarefaction curve illustrating 735

the decrease in extra proteins with each additional solubilization step. 736

737

Figure 5. Representation of C. difficile strain 630 genome highlighting genes 738

encoding spore proteome. a) Circular representation: the concentric circles represent the 739

following (from outside in):1+2, all CDSs (transcribed clockwise and counterclockwise); 740

3 locations of prophage (red), conjugative transposons (blue), carbohydrate utilization 741

(green) and cell wall loci (orange); 4+5 CDS that code for spore proteins (transcribed 742

clockwise and counterclockwise); 5 G+C content (plotted using a 10-kb window). b) 743

Linear representation of 27.8 kb region of 630 genome that contain the genes encoding 744

several spore proteins including a cysteine-rich, hypothetical protein (CD1067) that is 745

abundant in C. difficile spores. Red horizontal lines above CDS indicate those that 746

encode proteins found in mature C. difficile spores. Color coding for genes: dark blue, 747

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pathogenicity/adaptation; black, energy metabolism; red, information transfer; dark 748

green, surface-associated; cyan, degradation of large molecules; magenta, degradation of 749

small molecules; yellow, central/intermediary metabolism; pale green, unknown; pale 750

blue, regulators; orange, conserved hypothetical; brown, pseudogenes; pink, phage + IS 751

elements; gray, miscellaneous. 752

753

Figure 6. Representation of the distribution and abundance, by functional class, of 754

C. difficile 630 spore proteins. Absolute number of proteins (black bars, left axis) and 755

percentage of each functional class in spore relative to the total genome (grey bars, right 756

axis). Functional classes are described (34). 757

758

Figure 7. Comparative analysis of C. difficile 630 spore proteome to 11 clostridia 759

genomes. Summary of C. difficile spore protein functional classes a) shared among 760

clostridia, b) shared between C. difficile and C. bartlettii and c) unique to C. difficile. 761

762

supplemental Figure 1. Summary of comparative analysis of C. difficile 630 whole 763

genome and spore proteome to 11 clostridia and B. subtilis genomes. 3772 protein 764

sequences predicted from the C. difficile 630 genome (34) and 336 C. difficile spore 765

proteins were compared to the indicated genomes using Reciprocal FASTA analysis as 766

previously described (33). 767

768

supplemental Table 1. Summary of proteomic and genomic analysis of C. difficile 769

630 spores. Proteins present within C. difficile 630 spore grouped based on functional 770

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classification. Also included are summaries of protein solubilization and comparative 771

genome analysis. 772

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Figure 1. Visualization of pure C. difficile spores

a) b)sp

veg

ex

c) coat

cortex

membrane

core

ribosomes

exosporium

d)

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10 0

10 2

10 4

10 6

10 8

10 10

germinant added to BHI

10 0

10 2

10 4

10 6

10 8

0.5 1 3 24 0.5 1 3 24

hours at 60ºC hours at 70ºC

Water 70% EtOH 1% Virkon

10 0

10 2

10 4

10 6

10 8

treatment

a)

b)

c)

Figure 2. Pure C. difficile spores are viable and maintain resistant nature.

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Figure 3. Environmental C. difficile 630 spores are highly infective. a) Infective

dose curves of purified and fecal C. difficile 630 spores. b) C. difficile 630 establishes

low-level intestinal carriage in infected mice.

0

25

50

75

100

pure spores

fecal derived spores

environmental spores/cm 2

0 5 10 15 20 25 30 35 40 45 50100

102

104

106

108

10 10

clindamycin in H 2O

spore mediated transmission

days post-infection

a)

b)

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S p o r e sL D S b u f f e r / 2 � m e r c a p t o e t h a n o ls o l u b l ef r a c t i o n P e l l e tu r e a P e l l e t6 0 % F An e u t r a l iz e w i t h N H 3 3 H 2 OT C E Pt r y p t i c d ig e s tP e l l e tS D S � P A G E D i s c a r di n � g e l d ig e s tL C � M S / M S2 8 2 4 7 s p e c t r aM a s c o ts e a r c hv a l i d a t ee x t r a 7 1p r o t e i n sS D S � P A G Ei n � g e l d ig e s tL C � M S / M S3 9 8 3 0 s p e c t r aM a s c o ts e a r c hv a l i d a t e2 3 0 p r o t e i n s s o l u b l ef r a c t i o n s o l u b l ef r a c t i o nL C � M S / M SM a s c o ts e a r c hv a l i d a t e1 8 8 7 s p e c t r a

188

98

62

49

38

28

17146

0

5 01 0 01 5 02 0 02 5 03 0 0350N umb erofP rotei ns Id entifi ed e x t r a 3 2p r o t e i n sFigure 4. Proteomic analysis of C. difficile spores.

b)

a)

c)L D S s o l u b l e L D S + u r e a s o l u b l e L D S + u r e a s o l u b l e+ t r y p i c d i g e s t p e l l e tv e g s p o r e

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Figure 5. Representation of C. difficile strain 630 genome and spore genes

a)

p ep T C D 1 0 47C D 1 0 48C D 1 0 49 C D 1 0 5 0C D 1 0 5 1C D 1 0 5 2C D 1 0 5 3 b c d2 et f B 2 et f A 2 cr t 2 h b d t h l A 1 C D 1 0 6 0 C D 1 0 6 1a cp P C D 1 0 6 3C D 1 0 6 3 AC D 1 0 6 3 C c cp AC D 1 0 6 5 C D 1 0 6 6 C D 1 0 6 7C D 1 0 6 8 C D 1 0 6 9 C D 1 0 7 0 C D 1 0 7 1C D 1 0 7 2

1

b)

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Figure 6. Functional classification of C. difficile spore proteins.

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Figure 7. Comparative analysis of C. difficile spore genes to 11 clostridial genomes.

Conserved Clostridial Spore Proteome (88)

Gut-Dweller Spore Proteome (17)

C. difficile Unique Spore Proteome (29)

Proteases

Chaperones

Pathogenicity andAdaptation

Energy metabolism

Transcription, translationand DNA modification

Surface exposed proteins

Degradation of largemolecules

Degradation of smallmolecules

Central and intermediarymetabolism

Hypothetical proteins

Regulatory and signallingproteins

Conserved hypotheticalproteins

Pseudogenes

Mobile element genes

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