Quantitative Proteomic Analysis of the Heat Stress Response in Clostridium difficile Strain 630

Abstract

Clostridium difficile is a serious nosocomial pathogen whose prevalence worldwide is increasing. Postgenomic technologies can now be deployed to develop understanding of the evolution and diversity of this important human pathogen, yet little is known about the adaptive ability of C. difficile. We used iTRAQ labeling and 2D-LC-MS/MS driven proteomics to investigate the response of C. difficile 630 to a mild, but clinically relevant, heat stress. A statistically validated list of 447 proteins to which functional roles were assigned was generated, allowing reconstruction of central metabolic pathways including glycolysis, γ-aminobutyrate metabolism, and peptidoglycan biosynthesis. Some 49 proteins were significantly modulated under heat stress: classical heat shock proteins including GroEL, GroES, DnaK, Clp proteases, and HtpG were up-regulated in addition to several stress inducible rubrerythrins and proteins associated with protein modification, such as prolyl isomerases and proline racemase. The flagellar filament protein, FliC, was down-regulated, possibly as an energy conservation measure, as was the SecA1 preprotein translocase. The up-regulation of hydrogenases and various oxidoreductases suggests that electron flux across these pools of enzymes changes under heat stress. This work represents the first comparative proteomic analysis of the heat stress response in C. difficile strain 630, complementing the existing proteomics data sets and the single microarray comparative analysis of stress response. Thus we have a benchmark proteome for this pathogen, leading to a deeper understanding of its physiology and metabolism informed by the unique functional and adaptive processes used during a temperature upshift mimicking host pyrexia.

Secretome analysis of Clostridium difficile strains

“…CD630 that is well characterized and produces the main virulence factors toxin A and toxin B was used as reference strain. However, only a few studies described the proteome of C. difficile (Mukherjee et al 2002;Wright et al 2005;Lawley et al 2009;Jain et al 2010Jain et al , 2011. In all these analyses, only small numbers of proteins were detected and identified.…”

“…In all these analyses, only small numbers of proteins were detected and identified. Jain et al (2010) analyzed the insoluble subproteome of C. difficile, and Jain et al (2011) analyzed the complete proteome and characterized the heat stress response in C. difficile strain 630.…”

Proteomic Analysis and Label-Free Quantification of the Large Clostridium difficile Toxins

“…Proteomic examinations have been performed for C. difficile in which proteins released in vitro during high toxin production [7] were identified for strains 630 and VPI 10461. Additional studies characterizing the subproteomes of C. difficile reference strain 630, including a surface protein and insoluble protein fraction analysis [9, 10], spore protein identification [11], and a protein assessment associated with heat stress responses, have also been reported [12]. In an additional study, culture supernatants of C. difficile reference strain 630 were compared to two hypervirulent strains (CD196 and CDR20291), and five secreted proteins were identified exclusively in the supernatants of the hypervirulent strains [13].…”

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation

“…Thus, as in our previous work, all samples were analysed three separate times [40] resulting in increased overall peptide identifications. With a multiple injection workflow, the resolution of the MS becomes the limiting factor in proteome penetration.…”

“…Whilst these provide useful global snapshots of metabolic function, measurement of the adaptive response of the C. difficile proteome is much less advanced. Two recent reports describing iTRAQ-driven analysis of the organism's response to clinically relevant stress [40], exist, both complemented by analysis of the transcriptional programme under the same conditions [29], [42]. However, proteomes generated by isobaric labelling necessarily comprise only those proteins found in both experimental conditions, and yield few insights into those unique to each experimental condition [43].…”