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.
Highlights
Clostridium difficile, a Gram positive spore forming anaerobic bacterium, infects the human colonic epithelia causing diarrhoeal infections with symptoms including mild, self limiting diarrhoea with associated abdominal pain, cramping, and low grade fever
We wished in addition to establish some parameters regarding overall reproducibility of our GeLC/MS method by applying the exponentially modified protein abundance index (emPAI) workflow, which we have previously successfully applied to the comprehensive analysis of the soluble subproteome of Ochrobactrum anthropi at two distinct phases of growth [48]
Our systems biology data is consistent with the in vitro biochemistry and stoichiometry elucidated for this pathway – which we have identified in three independent proteomics investigations, thereby emphasising its importance during heat stress in C. difficile
Summary
Clostridium difficile, a Gram positive spore forming anaerobic bacterium, infects the human colonic epithelia causing diarrhoeal infections with symptoms including mild, self limiting diarrhoea with associated abdominal pain, cramping, and low grade fever (up to 40.6uC). The factors underlying CDI – including extended hospitalisation and the widespread administration of broad spectrum antibiotics – and the organism’s pathogenesis are well understood [2,3] and C. difficile is said to be most frequent cause of infectious bacterial diarrhoea in hospitals worldwide [4]. In addition to gastrointestinal disease, complications including build up of fluid in the peritoneal cavity and between the pleural layers of the lungs (ascites & pleural effusion, respectively), hepatic abscesses and renal failures have been reported [5] and worldwide, the cost of CDI is increasing annually [6,7]. The ‘hypervirulence’ of ribotype 027 strains has previously been attributed in part to increased sporulation, yet recent work has shown that ribotype 027 strains do not, sporulate more readily or at higher rates than other, non ribotype 027 strains [17]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
