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.

Tracking gene expression and oxidative damage of O2-stressed Clostridioides difficile by a multi-omics approach

“…Understanding the physiology of the pathogen and how it adapts to environmental changes is therefore a prerequisite for the development of novel therapies. In several published studies, omics-technologies have been employed to investigate the response and adaptation of C. difficile to stress and starvation [9][10][11]. With respect to the strictly anaerobic lifestyle of some human pathogenic bacteria, the presence of oxygen represents a major challenge [12].…”

The protein inventory of Clostridium difficile grown in complex and minimal medium

“…The first proteomics analysis carried out in C. difficile concentrated on specific subproteomes of the bacterium, that is, the cell envelope proteome , proteins of the spore outer layers , or secreted proteins in a comparative study of three different strains . Other groups performed proteomics analyses in order to unravel C. difficile ’s response to heat stress and the cationic antimicrobial peptide cathelicidin LL‐37 . Cafardi et al.…”

“…The first proteomics analysis carried out in C. difficile concentrated on specific subproteomes of the bacterium, that is, the cell envelope proteome [5], proteins of the spore outer layers [6][7][8], or secreted proteins in a comparative study of three different strains [9]. Other groups performed proteomics analyses in order to unravel C. difficile's response to heat stress [10,11] and the cationic antimicrobial peptide cathelicidin LL-37 [12]. Cafardi et al [13] and Pantaléon et al [14] analyzed the extracellular proteome, whereas the latter group also investigated surface proteins and biofilm matrix associated proteins to resolve the processes involved in biofilm formation and cell envelope properties of C. difficile cells growing in biofilms.…”

Vegetative Cell and Spore Proteomes ofClostridioides difficileshow finite differences and reveal potential biomarkers.

“…Eight Cwp family proteins are quantified: Cwp18 and 22 are identified in both morphotypes, with higher levels in spores, whereas Cwp2, 5, 6, 19, 84, and CwpV are identified in vegetative cells. Cwp22, a functional homologue of LD-transpeptidase (Ldt cd2 ), is an important spore protein that plays a role in peptidoglycan remodelling and confers resistance to β-lactam antibiotics (35, 36). CwpV promotes C. difficile aggregation and its strain-dependent structural variations may assist in evading the host antibody response (37) or to launch an anti-phage strategy (38).…”