Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile

Abstract

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficile cwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions.

Cwp22, a novel peptidoglycan cross‐linking enzyme, plays pleiotropic roles in Clostridioides difficile

“…In that study, Wydau‐Dematteis et al . (Wydau‐Dematteis et al ., ) proved that TcdE and bacteriolysis were coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions (works in BHIS not in TY). In this study, we also detected the increase of autolysis and cell permeability of the cwp22 mutant compared with the parent strain.…”

“…(Olling et al ., ) identified that the release of toxin from C. dificile 630Δ erm is not affected by the inactivation of the tcdE gene. Recently, it was identified that Cwp19 acts as a novel lytic tansglycosylase, which is involved in toxin release through stationary‐phase autolysis in C. difficile 630Δ erm (Wydau‐Dematteis et al ., ). In that study, Wydau‐Dematteis et al .…”

“…On the other hand, Olling et al (Olling et al, 2012) identified that the release of toxin from C. dificile 630Δerm is not affected by the inactivation of the tcdE gene. Recently, it was identified that Cwp19 acts as a novel lytic tansglycosylase, which is involved in toxin release through stationary-phase autolysis in C. difficile 630Δerm (Wydau-Dematteis et al, 2018). In that study, Wydau-Dematteis et al (Wydau-Dematteis et al, 2018) proved that TcdE and bacteriolysis were coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions (works in BHIS not in TY).…”

“…The role of the C. difficile S-layer in colonization, immunity, virulence and viability has been well-studied (Calabi et al, 2002;Pechine et al, 2005;Ausiello et al, 2006;Pechine et al, 2007;Kirk et al, 2017). Several other CWPs such as Cwp84, Cwp2, Cwp66, and CwpV have been investigated, and play important roles in host cell adhesion and immune system evasion during CDI (Waligora et al, 2001;Emerson et al, 2009;Kirby et al, 2009;Bradshaw et al, 2017;Wydau-Dematteis et al, 2018). Kirby et al (Kirby et al, 2009) identified a surface-associated protease, Cwp84, which cleaves SlpA precursor into mature form.…”

“…Cwp66, which works as an adhesin, and CwpV, which confers phase-variable phage infection resistance, were also characterized (Waligora et al, 2001;Emerson et al, 2009;Sekulovic et al, 2015). Recently, a new surface protein Cwp19, which is a novel lytic transglycosylase involved in stationary-phase autolysis and can affect toxin release in C. difficile, was also identified (Wydau-Dematteis et al, 2018). Biazzo et al (Biazzo et al, 2013) analysed 14 of the other 17 CWP genes scattered throughout the C. difficile genome, and identified the conserved DNA sequences and protein expression of Cwp13, CwpV, Cwp16, Cwp18, Cwp19, Cwp20, Cwp22, Cwp24 and Cwp25, suggesting that these CWPs may possess important functions in C. difficile.…”