The TcdE holin drives toxin secretion and virulence inClostridioides difficile

Abstract

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in ΔtcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in ΔtcdE mutants. Our findings confirm TcdE's critical functions in vivo for toxin secretion and C. difficile virulence.

A Streamlined Method to Obtain Biologically Active TcdA and TcdB Toxins from Clostridioides difficile

“…For instance, higher expression of tcdB could lead to higher expression of genes downstream of tcdB , such as tcdE . Since TcdE acts as a holin-like protein [ 37 ], its overexpression could increase cell lysis, therefore reducing protein yield for rTcdB. However, this hypothesis was beyond the scope of our work and was therefore not investigated further.…”

“…This could be due to the fact that the toxins are not secreted in our experimental conditions. However, recent studies have shown that toxin secretion varies across C. difficile strains [ 37 ]. Indeed, the 630 strain used in our work appears to be the strain for which toxin secretion was the lowest [ 37 ].…”

“…However, recent studies have shown that toxin secretion varies across C. difficile strains [ 37 ]. Indeed, the 630 strain used in our work appears to be the strain for which toxin secretion was the lowest [ 37 ]. Furthermore, toxins are secreted during the transition between exponential and stationary phases, while our work uses a short induction step during exponential growth.…”

“…Finally, secretion could also be hindered by the presence of a His-tag, preventing secretion. Authors interested in getting these toxins secreted could either use a strain background associated with high physiological secretion of toxins, such as the UK1 or VPI 10463 strains [ 37 ], or genetically modify the 630 strain to increase toxin secretion.…”

Exploring the Toxin-Mediated Mechanisms in Clostridioides difficile Infection

“…In the BI/NAP1/027 strain, the deletion of the tcdC gene has been implicated in the production of elevated levels of TcdA and TcdB [52,53]. The tcdE gene encodes the TcdE protein, which is a member of the class I family of holins and is believed to assist in toxin secretion [45,54]. Analysis of the PaLoc genome revealed the tcdL gene, which encodes the TcdL protein, an endolysin that interacts with TcdB and could be involved in toxin secretion [55,56].…”