Abstract
Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization.
Highlights
The Gram-positive obligate anaerobe Clostridioides difficile was first isolated in 1935 from neonates and identified as part of their natural intestinal microbiota
In order to determine the closest homolog of the virulence associated PrsA2 of L. monocytogenes (LmPrsA2), we performed an amino acid sequence alignment with Clustal Omega (Sievers et al, 2011)
We used the sequences of LmPrsA2 and the four putative parvulin-type PPIases from C. difficile, CD630_13570, CD630_15570, CD630_22630 as well as CD630_35000
Summary
The Gram-positive obligate anaerobe Clostridioides difficile was first isolated in 1935 from neonates and identified as part of their natural intestinal microbiota. Clostridioides difficile infection is mainly mediated by two enterotoxins (TcdA and TcdB) that are essential for developing the disease (Burke and Lamont, 2014) These toxins are taken up by endocytosis, translocate into the host cell cytosol and exert their activity by glycosylating and thereby inactivating small GTPases in human enterocytes. Apart from its enterotoxins, several other virulence factors that contribute to disease severity and host colonization have been described for C. difficile and analyzed to different extents These include, among others, the binary toxin CDT (C. difficile toxin) that is present in 5–6% of historic human isolates, extracellular proteases, surface layer proteins, several adhesins like a fibronectin binding protein (Fbp68) or a collagen binding protein (CbpA), flagella and type IV pili (Geric et al, 2004; Janoir, 2016; Ünal and Steinert, 2016; Péchiné et al, 2018). The arsenal of virulence factors and mechanisms that contribute to host colonization, disease outbreak and dissemination is still largely unexplored
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