title: Semiquantitative analysis of clinical heat stress in Clostridium difficile strain 630 using a GeLC/MS workflow with emPAI quantitation.
abstract: Clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 C. difficile proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.
authors.affiliation: Northern Ireland Centre for Food and Health (NICHE), School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, Northern Ireland, United Kingdom.
authors.forename: Nigel G
authors.initials: NG
authors.lastname: Ternan
authors.affiliation: Northern Ireland Centre for Food and Health (NICHE), School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, Northern Ireland, United Kingdom.
authors.forename: Shailesh
authors.initials: S
authors.lastname: Jain
authors.affiliation: School of Medicine, University of Manchester, Manchester, Greater Manchester, United Kingdom.
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.affiliation: Northern Ireland Centre for Food and Health (NICHE), School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, Northern Ireland, United Kingdom.
authors.forename: Geoff
authors.initials: G
authors.lastname: McMullan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D016360:Clostridioides difficile / Q000235:genetics* / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D006358:Hot Temperature; D020543:Proteome / Q000235:genetics* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry; D015854:Up-Regulation / Q000235:genetics; D057188:Workflow
chemical_list: D001426:Bacterial Proteins; D020543:Proteome