Abstract
BackgroundClostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. The epidemic successful strains are also associated with severe clinical features of CDI. Therefore, a proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and label-free quantification (LFQ) approach.ResultsThe comparative semi-quantitative analyses enabled investigation of a total of 662 proteins. Both hierarchical clustering and principal component analysis (PCA) created eight distinctive groups. From these quantifiable proteins, 27 were significantly increased in functional annotations. Among them, several known factors connected with virulence were identified, such as toxin A, B, binary toxin, flagellar proteins, and proteins associated with Pro–Pro endopeptidase (PPEP-1) functional complex. Comparative analysis of protein expression showed a higher expression or unique expression of proteins linked to pathogenicity or iron metabolism in RTs 027 and 176 supporting their genetic relatedness and clinical importance at the proteomic level. Moreover, the absence of putative nitroreductase and the abundance of the Abc-type fe3+ transport system protein were observed as biomarkers for the RTs possessing binary toxin genes (027, 176 and 078). Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic role of the assembly in CDI. Finally, the histidine synthesis pathway regulating protein complex HisG/HisZ was observed only in isolates possessing the genes for toxin A and B.ConclusionsThis study showed the applicability of the LFQ approach and provided the first semi-quantitative insight into the proteomes released from in vitro cultured panel of eight RTs. The observed differences pointed to a new direction for studies focused on the elucidation of the mechanisms underlining the CDI nature.
Highlights
Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon
Comparison of proteomes of individual RTs To assess the applicability of the labelfree quantification (LFQ) approach, we examined the similarity of individual proteomes using hierarchical clustering and principal component analysis
The unlimited number of compared groups in the LFQ technique enabled the semi-quantitative investigation of the proteomes released from in vitro cultured panel of eight C. difficile isolates and the applicability of this workflow was shown by hierarchical clustering and principal component analysis (PCA); both methods generated eight distinctive groups encompassing each biological triplicate of RTs analyzed
Summary
Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. A proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and labelfree quantification (LFQ) approach. Toxin-producing strains of C. difficile can cause infection (CDI) manifested by diarrhea, pseudomembranous colitis, or severe form, toxic megacolon. The spread of certain C. difficile PCR ribotypes in health-care setting has been reported and the global increasing trend of CDI incidence is unfavorable [1]. Previous proteomic studies were based on investigation of either the whole cell lysates [4, 5], or culture supernatants representing possible secretome in vitro [2, 6]. For comparative proteomic analyses difference gel electrophoresis (DIGE) [4] or isotopic labelling of selected proteins [7] were used
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