Article pubs.acs.org/jpr
Proteomic Comparison of Historic and Recently Emerged Hypervirulent Clostridium dif ficile Strains Jenn-Wei Chen,†,‡ Joy Scaria,†,‡ Chunhong Mao,§ Bruno Sobral,§ Sheng Zhang,‡ Trevor Lawley,⊥ and Yung-Fu Chang*,† †
Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, New York 14853, United States Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, Virginia 24061, United States ‡ Proteomics and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York 14853, United States ⊥ Microbial Pathogenesis Laboratory, The Wellcome Trust Sanger Institute, Hinxton, United Kingdom §
S Supporting Information *
ABSTRACT: Clostridium dif f icile in recent years has undergone rapid evolution and has emerged as a serious human pathogen. Proteomic approaches can improve the understanding of the diversity of this important pathogen, especially in comparing the adaptive ability of different C. dif f icile strains. In this study, TMT labeling and nanoLC−MS/MS driven proteomics were used to investigate the responses of four C. dif f icile strains to nutrient shift and osmotic shock. We detected 126 and 67 differentially expressed proteins in at least one strain under nutrition shift and osmotic shock, respectively. During nutrient shift, several components of the phosphotransferase system (PTS) were found to be differentially expressed, which indicated that the carbon catabolite repression (CCR) was relieved to allow the expression of enzymes and transporters responsible for the utilization of alternate carbon sources. Some classical osmotic shock associated proteins, such as GroEL, RecA, CspG, and CspF, and other stress proteins such as PurG and SerA were detected during osmotic shock. Furthermore, the recently emerged strains were found to contain a more robust gene network in response to both stress conditions. This work represents the first comparative proteomic analysis of historic and recently emerged hypervirulent C. dif f icile strains, complementing the previously published proteomics studies utilizing only one reference strain. KEYWORDS: Clostridium dif f icile, comparative proteomic analysis
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INTRODUCTION Clostridium dif f icile is the leading cause of nosocomial diarrhea in North America and Europe.1 C. dif f icile infection (CDI) is now increasingly being reported from all over the world.1 In recent years, CDI has been reported in the general community as well.2 The complications arising from CDI can range from mild self-limiting diarrhea to toxic megacolon, intestinal perforation, or death.3 CDI rate in the United States estimated based on hospital discharge diagnosis between the years 2000 and 2008 reveals that it has nearly tripled since 2000.4 A conservative estimate of economic costs associated with CDI in the United States is projected to be $3.2 billion annually.5 One of the reasons for this resurgence in the CDI rate is the emergence of highly divergent C. dif f icile strains.6,7 A major portion of CDI has been caused by a recently emerged hypervirulent strain belonging to PCR ribotype 027/pulse-field type NAP1 (027/NAP1) and is associated with outbreaks and severe mortalities.8,9 Comparative genomic analysis of several nonhypervirulent and hypervirulent C. dif ficile strains has revealed that there is massive genome diversity among these strains.10−13 Genome © 2013 American Chemical Society
scale evolutionary analysis of C. dif f icile strains isolated between 1980s and 2007 has demonstrated the existence of large scale genome variation that extends to even to the core genome.11 In this perspective, virulent strains isolated in the 1980s are considered historic while hypervirulent epidemic strains isolated after 2000 are considered recently emerged. Previously, proteomics and transcriptomic studies in C. dif f icile have been carried out to analyze the cell surface proteins,14 spore associated proteins,15 and heat shock response.16−18 Despite the considerable genome variation, all of these investigations on C. dif ficile have been carried out using historic reference strain CD630.15−18 This is partly because CD630 was the first strain that was sequenced using the Sanger sequencing method.19 This strain was isolated in 1982 from a patient with pesudomembraneous colitis from Zurich, Switzerland.19 However, in the past few years, several more historic as well as newly emerged C. dif f icile strains have been sequenced using new generation DNA sequencing methods.10−13 To understand Received: August 8, 2012 Published: January 8, 2013 1151
dx.doi.org/10.1021/pr3007528 | J. Proteome Res. 2013, 12, 1151−1161
Journal of Proteome Research
Article
how the historic and recently emerged C. dif f icile strains respond to the environmental changes, in this study we have compared the proteome of multiple sequenced C. dif f icile strains after subjecting them to physiologically relevant in vitro stress conditions. The strains in this study include two virulent strains isolated in the 1980s (CD630 and CD196) and two hypervirulent strains isolated after 2000 (R20291 and QCD32g58). Our results show that when compared to historic isolates, the recent hypervirulent strains have a more robust stress response that might enable better colonization and dissemination of these strains.
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followed by ImageQuant Software version 5.2 (GE Healthcare). A total of 50 μg protein of each sample was reduced with 11.9 mM tris(2-carboxyethyl)phosphine for 1 h at 56 °C, alkylated with 20 mM iodoacetamide for 30 min in the dark and then quenched by additional of 20 mM Dithiothreitol (DTT). Each sample was digested with 5 μg of sequencing grade trypsin (Promega, Madison, MI) overnight at 37 °C. The TMT 6-plex labels (dried powder) were reconstituted with 41 μL of anhydrous ACN prior to labeling and added with 1:1 ratio to each of the tryptic digest samples for labeling over 1 h at room temperature. The peptides were mixed with each respective tag as follows: two replicates of control group, TMT126 and TMT127; two replicates of nutrient shift group, TMT128 and TMT129; two replicates of osmotic shock group, TMT130 and TMT131. After checking label incorporation by MALDI-TOF/ TOF 4700 (Applied Biosystems, Foster City, CA) by randomly choosing 5 different peptide ions from each sample, the six samples were pooled, evaporated to dryness and subjected to cation exchange chromatography using a PolyLC strong cationexchange cartridge (PolyLC Inc. Columbia, MD). The SCX cartridge (10 mm id × 14 mm) is PolySULFOETHYL Aspartamide material with particle size at 12 μm and 300 Å pore size The pooled TMT labeled tryptic peptides were then reconstituted with 3.0 mL of loading buffer (10 mM potassium phosphate pH 3.0, 25% ACN). The pH of the sample was adjusted to 3.0 with formic acid prior to cartridge separation. After conditioning of the Strong-cation exchange (SCX) cartridge with loading buffer, the sample (∼300 μg) was loaded and washed with an additional 2.0 mL of loading buffer. The peptides were eluted in one step by 1.0 mL of loading buffer containing 500 mM KCl. Desalting of SCX fractions was carried out using solid phase extraction (SPE) on Sep-Pak Cartridges (Waters, Milford, MA) and the eluted tryptic peptides were evaporated to dryness, and ready for the first dimensional LC fractionation via a high pH reverse phase chromatography as described below.
EXPERIMENTAL SECTION
Bacterial Culture
All experiments were conducted in a Bactron IV anaerobic chamber (Shel Lab, Cornelius, OR) that was filled and purged with an anaerobic gas mixture (10% CO2, 85% N2, 5% H2). The chamber contained a catalyst, which removes any trace amounts of oxygen. All materials used in the anaerobic chamber were prereduced before use. Spores of C. dif f icile strains CD630, CD196, QCD32g58 and R20291 were streaked on brain-heart infusion (BHI) agar plates containing 0.1% L-cysteine and taurocholate. The plates were incubated overnight at 37 °C. Single colonies from these plates were then used inoculate prereduced BHI broth and were incubated at 37 °C overnight. Fresh BHI broth was then inoculated by transferring 1% overnight culture. Cultures were incubated at 37 °C until the OD600 reached between 0.4 and 0.5. Bacteria were then collected by centrifugation at 2000× g for 5 min. These cells were then shifted to two physiologically relevant in vitro conditions. First was nutrient change by shifting to an equal volume of Basal defined medium (BDM)20 with supplementation of 0.5% sucrose. This shift was designed to induce multiple nutritional changes and to analyze the impact of those changes in C. dif f icile pathways. Second was osmotic shock by shifting to an equal volume of BHI supplemented with 1.5% NaCl. The same amount of cells was transferred to fresh BHI as control group. After the incubation for 1 h at 37 °C, the bacteria were collected by centrifugation at 2000× g for 10 min. All experiments were performed as separate biological replicates.
High pH Reverse Phase (hpRP) Fractionation
The hpRP chromatography was carried out using a Dionex UltiMate 3000 HPLC system with the built-in micro fraction collection option in its autosampler and UV detection (Sunnyvale, CA) as reported previously.21 The TMT 6-plex tagged tryptic peptides were reconstituted in buffer A (20 mM ammonium formate pH 9.5 in water), and loaded onto an XTerra MS C18 column (3.5 μm, 2.1 × 150 mm) from Waters, (Milford, MA) with 20 mM ammonium formate (NH4FA), pH 9.5 as buffer A and 80% ACN/20% 20 mM NH4FA as buffer B. The LC was performed using a gradient from 10 to 45% of buffer B in 30 min at a flow rate 200 μL/min. Forty-eight fractions were collected at 1 min intervals and pooled into a total of 12 fractions based on the UV absorbance at 214 nm and with multiple fraction concatenation strategy.22 This concatenation strategy is basically a “retention time multiplexing” approach through pooling disparate first dimensional HpRP fractions prior to the second dimension nanoLC−MS/MS analysis without any appreciable degradation in chromatographic resolution or reduction in peptide identifications compared to the individually analyzed fractions, as first reported by Dr. Smith’s group.23 All of the fractions were dried and reconstituted in 150 μL of 2% ACN/0.5% Formic acid for nanoLC−MS/MS analysis.
Protein Extraction, Digestion and TMT Labeling
The bacterial pellet was washed with PBS and subjected to centrifugation at 2000× g for 10 min. The bacterial protein was then extracted with FOCUS Bacterial Proteome kit (GBiosciences, St Louis, MO) according to the manufacturer’s instruction. The protein sample in FPS buffer was precipitated by adding 4 volumes of ice-cold acetone and incubating for 3 h at −20 °C. Then the protein pellet was collected by centrifugation at 13000× g for 15 min. Further processing of the proteins was then performed according to Thermo Scientific’s TMT Mass Tagging Kits and Reagents protocol (http://www.piercenet.com/instructions/2162073.pdf) with a slight modification. Briefly, protein pellets were resuspended and denatured in a final concentration of 200 mM triethylammoniumbicarbonate, 6 M Guanidine-HCl and 0.1% SDS, pH 8.5 at 56 °C for 1 h. The protein concentration for each sample was determined by Bradford assay using BSA as the calibrant, and further quantified by running on a precast NOVEX 12% Tris/Glycine mini-gel (Invitrogen, Carlsbad, CA) along with a series of amounts of E. coli lysates (2, 5, 10, 20 μg/ lane). The SDS gel was visualized with colloidal Coomassie blue stain (Invitrogen), imaged by Typhoon 9400 scanner 1152
dx.doi.org/10.1021/pr3007528 | J. Proteome Res. 2013, 12, 1151−1161
Journal of Proteome Research
Article
Nanoscale Reverse Phase Chromatography and Tandem Mass Spectrometry (nanoLC−MS/MS)
database is generated and tested for raw spectra along with the real database. To reduce the probability of false peptide identification, the significant scores at 99% confidence interval for the peptides defined by a Mascot probability analysis (www. matrixscience.com/help/scoring_help.html#PBM) greater than “identity” were used as a filter along with 1.5 or
