Abstract
The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.
Highlights
The multi-resistant Gram-positive obligate anaerobe Clostridioides (Clostridium) difficile is the main nosocomial bacterial agent of antibiotic treatment associated diarrhea (Hopkins and Wilson, 2018)
Our results show an unprecedented relationship between bacterial cysteine metabolism and CdPpiB with far reaching implications on virulence
An exceptional high degree of conservation was observed for amino acid residues involved in PPIase activity (Figure 1). This gene was selected for destruction by ClosTron, which resulted in the generation of a ppiB mutant in the C. difficile str. 630 erm background
Summary
The multi-resistant Gram-positive obligate anaerobe Clostridioides (Clostridium) difficile is the main nosocomial bacterial agent of antibiotic treatment associated diarrhea (Hopkins and Wilson, 2018). High recurrence rates, especially after treatment with vancomycin or metronidazole, make this disease difficult to manage (Mullane, 2014; Hopkins and Wilson, 2018). CDI manifests in form of pseudomembranous colitis, which is a strong inflammation of the large intestine. This is caused by massive tissue destruction and leukocytosis due to the production of two large glycosylating toxins, TcdA and TcdB. These exert their cytotoxic activity by inactivating small GTPases and disturbing the actin cytoskeleton dynamics leading to disruption of the gut epithelial barrier (Barbut et al, 2007; Rupnik et al, 2009; Chandrasekaran and Lacy, 2017)
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