PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator

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.

The Transcriptional Regulator Lrp Contributes to Toxin Expression, Sporulation, and Swimming Motility in Clostridium difficile

“…The severity of infection was clearly indicated in the mice infected with the R20291 lrp mutant strain, attributed to smaller cecum size and less well-formed feces, as well as more extensive necrosis and inflammation, as revealed by histological examination. It has been suggested in the past that clindamycin administration prior to challenge with C. difficile select for Clostron-based mutants bearing the ermB cassette, although other studies in which Clostron-based mutants with reduced virulence in vivo have also been reported (Ünal et al, 2018; Zhu et al, 2019). However, for further clarification, we are in the process of obtaining a markerless lrp mutant using the recently developed RiboCas system (Cañadas et al, 2019).…”

Impacts of the Type I Toxin–Antitoxin System, SprG1/SprF1, on Staphylococcus aureus Gene Expression

“…PpiB (also referred to as PPIase) catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, thus accelerating protein folding. PPIases are required for the correct folding and subsequent activity of secreted virulence factors in a number of bacterial pathogens [ 34 , 35 ]. Another protein downregulated in S. aureus N315 ΔsprG1/ΔsprF1 + pCN35Ω sprF1 , in the intra- and extracellular proteomes, is PdhB.…”

Role of protein repair enzymes in oxidative stress survival and virulence of Salmonella

“…The role of PPIase in bacterial virulence is mostly explained by its ability to facilitate proper folding of secreted proteins, adhesins, and other virulence factors (Hermans et al 2006;Purdy et al 2007;Alonzo and Freitag, 2010;Behrens-Kneip, 2010;Forster et al 2011). In Streptococcus suis, Listeria monocytogenes, and Clostridioides difficile, PPIases are required for resistance against several stresses including thermal, oxidative, and acid stresses, thereby contributing to virulence in mice (Bigot et al 2006;Wu et al 2011;Ünal et al 2018). PPIase gene deletion strains of E. coli and Yersinia pseudotuberculosis showed defective adherence to and invasion of host cells (Justice et al 2006;Obi and Francis, 2013) and virulence in mice (Hermans et al 2006;Cron et al 2009).…”