Abstract
Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, anaerobe, spore-forming pathogen, which causes drug-induced diseases in hospitals worldwide. A detailed analysis of the proteome may provide new targets for drug development or therapeutic strategies to combat this pathogen. The application of metabolic labeling (ML) would allow for accurate quantification of significant differences in protein abundance, even in the case of very small changes. Additionally, it would be possible to perform more accurate studies of the membrane or surface proteomes, which usually require elaborated sample preparation. Such studies are therefore prone to higher standard deviations during the quantification. The implementation of ML strategies for C. difficile is complicated due to the lack in arginine and lysine auxotrophy as well as the Stickland dominated metabolism of this anaerobic pathogen. Hence, quantitative proteome analyses could only be carried out by label free or chemical labeling methods so far. In this paper, a ML approach for C. difficile is described. A cultivation procedure with 15N-labeled media for strain 630Δerm was established achieving an incorporation rate higher than 97%. In a proof-of-principle experiment, the performance of the ML approach in C. difficile was tested. The proteome data of the cytosolic subproteome of C. difficile cells grown in complex medium as well as two minimal media in the late exponential and early stationary growth phase obtained via ML were compared with two label free relative quantification approaches (NSAF and LFQ). The numbers of identified proteins were comparable within the three approaches, whereas the number of quantified proteins were between 1,110 (ML) and 1,861 (LFQ) proteins. A hierarchical clustering showed clearly separated clusters for the different conditions and a small tree height with ML approach. Furthermore, it was shown that the quantification based on ML revealed significant altered proteins with small fold changes compared to the label free approaches. The quantification based on ML was accurate, reproducible, and even more sensitive compared to label free quantification strategies.
Highlights
Clostridioides difficile (Lawson et al, 2016), formerly known as Clostridium difficile (Hall and O’Toole, 1935), is an ubiquitous, obligate anaerobic, spore forming, Gram-positive bacterium with a close relation to the Peptostreptococcaceae family (Collins et al, 1994; Yutin and Galperin, 2013)
A number of quantitative proteome analyses were published for C. difficile based on label free quantification (LFQ) (Chilton et al, 2014; Otto et al, 2016; Dresler et al, 2017) or chemical labeling (Jain et al, 2011; Chen et al, 2013; Chong et al, 2014; Pettit et al, 2014; Charlton et al, 2015)
The introduction of a stable isotope into proteins by application of metabolic labeling (ML) enables for accurate quantification of minor changes even after complex and error-prone sample preparation workflows, e.g., by subcellular fractionation (Bantscheff et al, 2007)
Summary
Clostridioides difficile (Lawson et al, 2016), formerly known as Clostridium difficile (Hall and O’Toole, 1935), is an ubiquitous, obligate anaerobic, spore forming, Gram-positive bacterium with a close relation to the Peptostreptococcaceae family (Collins et al, 1994; Yutin and Galperin, 2013). C. difficile is the leading cause of healthcare-associated infective diarrhea (Freeman et al, 2010) with approximately 500,000 patients in the United States (Lessa et al, 2015) and 124,000 patients in the European Union in 2011 (Smits et al, 2016). In addition to a variety of other studies, the publications on cell wall associated proteins and proteins of the spore layers are interesting. Cell wall associated proteins of the pathogen interact with the host and the microbiota and could be potential targets for the immune system or antimicrobial agents while spores play an important role for the infection as well as during spreading processes and antibiotic resistance. Proteins of different spore layers involved in the spore coat morphogenesis, in the attachment to surfaces, or proteins with a possible role in spore resistance or germination were identified (Abhyankar et al, 2013; Swarge et al, 2018)
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