Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different toxinotypes using small samples has not yet been reported. In the present study, we quantify C. difficile toxins in <0.1？mL of culture filtrate by quantitative label-free mass spectrometry (MS) using data-independent analysis (MSE). In addition, analyses of both purified TcdA and TcdB as well as a standard culture filtrate were performed using gel-based and gel-independent proteomic platforms. Gel-based proteomic analysis was then used to generate basic information on toxin integrity and provided sequence confirmation. Gel-independent in-solution digestion of both toxins using five different proteolytic enzymes with MS analysis generated broad amino acid sequence coverage (91% for TcdA and 95% for TcdB). Proteomic analysis of a culture filtrate identified a total of 101 proteins, among them TcdA, TcdB, and S-layer proteins. 1. Introduction Clostridium difficile is a gram-positive, anaerobic, spore-forming, rod-shaped bacterium that can produce at least three toxins including two Rho GTPase-glucosylating toxins (TcdA and TcdB) and the binary C. difficile transferase (CDT) toxin. The organism can cause C. difficile infection (CDI) in humans and animals. C. difficile is considered an important cause of healthcare-associated infection in humans and is the leading cause of antibiotic-associated diarrhea in hospitals worldwide [1, 2]. CDIs are toxin-mediated illnesses that range from mild diarrhea to fulminant pseudomembranous colitis and toxic megacolon, which may result in death [3]. Laboratorial confirmation of CDI needs to be rapidly performed. The methods include detection of C. difficile through cultivation, detection of Tcd A and Tcd B in stool samples using immunoassay and DNA-based methods [2]. Reflecting its changing epidemiology, the incidence and severity of CDI have increased significantly in the past ten years. Among the possible causes of increasing morbidity is the emergence of strains considered to be more virulent [4]. These strains produce greater amounts of toxin than reference strains and are highly transmissible due to their greater sporulation capacity [4]. One example is the rapid emergence of the highly virulent clone-designated BI/NAP1/027 in multiple countries [5]. Additionally, it is estimated that there are 500,000 cases of CDI per year in US
References
[1]
G. P. Carter, J. I. Rood, and D. Lyras, “The role of toxin A and toxin B in Clostridium difficile-associated disease: past and present perspectives,” Gut Microbes, vol. 1, no. 1, pp. 58–64, 2010.
[2]
M. Rupnik, M. H. Wilcox, and D. N. Gerding, “Clostridium difficile infection: new developments in epidemiology and pathogenesis,” Nature Reviews Microbiology, vol. 7, no. 7, pp. 526–536, 2009.
[3]
C. V. Gould and L. C. McDonald, “Bench-to-bedside review: Clostridium difficile colitis,” Critical Care, vol. 12, no. 1, p. 203, 2008.
[4]
M. Merrigan, A. Venugopal, M. Mallozzi et al., “Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production,” Journal of Bacteriology, vol. 192, no. 19, pp. 4904–4911, 2010.
[5]
M. Warny, J. Pepin, A. Fang et al., “Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe,” Lancet, vol. 366, no. 9491, pp. 1079–1084, 2005.
[6]
S. Karlsson, L. G. Burman, and T. ？kerlund, “Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism,” Microbiology, vol. 154, no. 11, pp. 3430–3436, 2008.
[7]
K. Mukherjee, S. Karlsson, L. G. Burman, and T. ？kerlund, “Proteins released during high toxin production in Clostridium difficile,” Microbiology, vol. 148, no. 7, pp. 2245–2253, 2002.
[8]
P. Vohra and I. R. Poxton, “Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile,” Microbiology, vol. 157, no. 5, pp. 1343–1353, 2011.
[9]
A. Wright, D. Drudy, L. Kyne, K. Brown, and N. F. Fairweather, “Immunoreactive cell wall proteins of Clostridium difficile identified by human sera,” Journal of Medical Microbiology, vol. 57, no. 6, pp. 750–756, 2008.
[10]
A. Wright, R. Wait, S. Begum et al., “Proteomic analysis of cell surface proteins from Clostridium difficile,” Proteomics, vol. 5, no. 9, pp. 2443–2452, 2005.
[11]
T. D. Lawley, N. J. Croucher, L. Yu et al., “Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores,” Journal of Bacteriology, vol. 191, no. 17, pp. 5377–5386, 2009.
[12]
S. Jain, C. Graham, R. L. J. Graham, G. McMullan, and N. G. Ternan, “Quantitative proteomic analysis of the heat stress response in Clostridium difficile strain 630,” Journal of Proteome Research, vol. 10, no. 9, pp. 3880–3890, 2011.
[13]
A. Boetzkes, K. W. Felkel, J. Zeiser, N. Jochim, I. Just, and A. Pich, “Secretome analysis of Clostridium difficile strains,” Archives of Microbiology, 2012.
[14]
J. R. Barr, V. L. Maggio, D. G. Patterson Jr. et al., “Isotope dilution—mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I,” Clinical Chemistry, vol. 42, no. 10, pp. 1676–1682, 1996.
[15]
B. Domon and R. Aebersold, “Options and considerations when selecting a quantitative proteomics strategy,” Nature Biotechnology, vol. 28, no. 7, pp. 710–721, 2010.
[16]
S. Pan, R. Aebersold, R. Chen et al., “Mass spectrometry based targeted protein quantification: methods and applications,” Journal of Proteome Research, vol. 8, no. 2, pp. 787–797, 2009.
[17]
W. Zhu, J. W. Smith, and C. M. Huang, “Mass spectrometry-based label-free quantitative proteomics,” Journal of Biomedicine and Biotechnology, vol. 2010, Article ID 840518, 6 pages, 2010.
[18]
S. J. Geromanos, J. P. C. Vissers, J. C. Silva et al., “The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MS,” Proteomics, vol. 9, no. 6, pp. 1683–1695, 2009.
[19]
J. C. Silva, R. Denny, C. Dorschel et al., “Simultaneous qualitative and quantitative analysis of the Escherichia coli proteome: a sweet tale,” Molecular and Cellular Proteomics, vol. 5, no. 4, pp. 589–607, 2006.
[20]
A. E. Boyer, M. Gallegos-Candela, R. C. Lins et al., “Quantitative mass spectrometry for bacterial protein toxins—a sensitive, specific, high-throughput tool for detection and diagnosis,” Molecules, vol. 16, no. 3, pp. 2391–2413, 2011.
[21]
H. Moura, R. R. Terilli, A. R. Woolfitt et al., “Studies on botulinum neurotoxins type/C1 and mosaic/DC using Endopep-MS and proteomics,” FEMS Immunology and Medical Microbiology, vol. 61, no. 3, pp. 288–300, 2011.
[22]
R. R. Terilli, H. Moura, A. R. Woolfitt, J. Rees, D. M. Schieltz, and J. R. Barr, “A historical and proteomic analysis of botulinum neurotoxin type/G,” BMC Microbiology, vol. 11, article 232, 2011.
[23]
R. West, J. Whitmon, Y. M. Williamson et al., “A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms,” Journal of Proteomics, vol. 75, no. 6, pp. 1966–1972, 2012.
[24]
N. Y. Yu, J. R. Wagner, M. R. Laird et al., “PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes,” Bioinformatics, vol. 26, no. 13, pp. 1608–1615, 2010.
[25]
R. G. Biringer, H. Amato, M. G. Harrington, A. N. Fonteh, J. N. Riggins, and A. F. R. Hühmer, “Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS,” Briefings in Functional Genomics and Proteomics, vol. 5, no. 2, pp. 144–153, 2006.
[26]
F. Fischer and A. Poetsch, “Protein cleavage strategies for an improved analysis of the membrane proteome,” Proteome Science, vol. 4, article 2, 2006.
[27]
D. L. Swaney, C. D. Wenger, and J. J. Coon, “Value of using multiple proteases for large-scale mass spectrometry-based proteomics,” Journal of Proteome Research, vol. 9, no. 3, pp. 1323–1329, 2010.
[28]
J. E. Blake, F. Mitsikosta, and M. A. Metcalfe, “Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants,” Journal of Medical Microbiology, vol. 53, no. 3, pp. 197–205, 2004.
[29]
G. Yang, B. Zhou, J. Wang et al., “Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium,” BMC Microbiology, vol. 8, article 192, 2008.
[30]
T. ？kerlund, B. Svenungsson, ？. Lagergren, and L. G. Burman, “Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates,” Journal of Clinical Microbiology, vol. 44, no. 2, pp. 353–358, 2006.
[31]
Y. Levin, E. Hradetzky, and S. Bahn, “Quantification of proteins using data-independent analysis (MSE) in simple and complex samples: a systematic evaluation,” Proteomics, vol. 11, no. 16, pp. 3273–3287, 2011.
[32]
R. A. Stabler, M. He, L. Dawson et al., “Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium,” Genome Biology, vol. 10, no. 9, article R102, 2009.
