Comprehensive redox profiling of the thiol proteome of Clostridium difficile

The strictly anaerobic bacterium C. difficile has become one of the most problematic hospital acquired pathogens and a major burden for health care systems. Although antibiotics work effectively in most C. difficile infections (CDIs), their detrimental effect on the intestinal microbiome paves the way for recurrent episodes of CDI. To develop alternative, non-antibiotics-based treatment strategies, deeper knowledge on the physiology of C. difficile, stress adaptation mechanisms and regulation of virulence factors is mandatory. Focus of this work was to tackle the thiol proteome of C. difficile and its stress-induced alterations, since recent research reported the amino acid cysteine to play a central role in the metabolism of the pathogen. We developed a novel cysteine labeling approach to determine the redox state of protein thiols on a global scale. Applicability of the technique was demonstrated by inducing disulfide stress using the chemical diamide. The method can be transferred to any kind of redox challenge and was applied in this work to assess the effect of bile acids on the thiol proteome of C. difficile. We present redox-quantification of more than 1,500 thiol peptides and discuss the general difficulty of redox analyses of peptides possessing more than a single cysteine residue. The presented method will be especially useful when not only redox status shall be determined, but information on protein quantity is needed as well. Not least, our comprehensive dataset reveals protein cysteine sites particularly susceptible to oxidation and builds a groundwork for redox proteomics studies in C. difficile.