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.
Highlights
Clostridioides difficile is an opportunistic human pathogen that causes antibiotic-associated diarrhea (Schäffler and Breitrück, 2018)
In contrast to phage holins, an additional isoform with an N-terminal extension evolved in C. difficile which inhibits lysis in the absence of transported substrates, i.e., in the absence of an endolysin (Figure 4)
The finding that the N-terminal extension evolved most likely in PaLocencoded TcdE suggests that it is somehow related to toxins and their secretion
Summary
Clostridioides difficile is an opportunistic human pathogen that causes antibiotic-associated diarrhea (Schäffler and Breitrück, 2018). In the most recent studies on TcdE, the presence of differential translational start codons came into the focus (Govind and Dupuy, 2012; Olling et al, 2012; Govind et al, 2015), as previous studies on lambda phage holins showed a regulatory function of the co-existence of holin isoforms with close-by but distinct translational starts (Bläsi et al, 1990; Chang et al, 1995; Barenboim et al, 1999) In this well-studied case, only the short isoform is lytic, whereas the isoform extended by two residues is not lytic and believed to serve as antiholin whose production regulates the timing of lysis (Barenboim et al, 1999). TcdE could be shown to be able to transport endolysins (Govind and Dupuy, 2012) and nothing is known about recognitions or interactions that could mediate a toxin specificity
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