Evidence for an Adaptation of a Phage-Derived Holin/Endolysin System to Toxin Transport in Clostridioides difficile

Abstract

The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent transport.

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridium sordellii

“…This intermediary protein may come in the form of an endolysin that remodels the peptidoglycan to allow the transit of such a large protein, as has been suggested in S. marcescens and Salmonella Typhi [30,38,42]. This hypothesis is consistent with the presence of endolysin-encoding genes in the PaLoc of P. sordellii and in some strains of C. difficile [37,43]. In this model, the specificity of release for TcsL that occurs in the presence of TcsE (Figure 5, Table 1) would be indirect, with TcsE required to specifically translocate an endolysin required to act and then facilitate the translocation of TcsL across the P. sordellii cell wall.…”

Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation

“…However, C difficile-specific depletion, such as that achieved by treatment with C difficile-associated phage-derived endolysins, is effective against CDI. 21,24,25 Therefore, in addition to regulating Proteobacteria expansion, a characteristic of chronic intestinal inflammation during rCDI, the elimination of C difficile might be required.…”

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

“…In contrast, the presence of ArrA antiholin homologs without TeaA holin homologs raises the question: what are antiholins doing on their own? Perhaps they have evolved functionality with holins divergent enough to no longer be considered homologous to TeaA under our search parameters, they had previous functions before evolving to moderate lysis timing, or they have been coopted for divergent functions much like holins have been (47,48).…”