A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridiumsordellii.

Callum J Vidor,Audrey Hamiot,Dena Lyras,Rommel A Mathias,Jessica Wisniewski,Bruno Dupuy,Milena Awad

doi:10.3390/toxins14020124

Abstract

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs.

Highlights

Introduction distributed under the terms andProtein secretion, defined as the transport of proteins from the cell cytoplasm to the extracellular milieu or into a target cell [1], is a critically important process in all bacteria
To determine the roles of the Pathogenicity Locus (PaLoc)-associated holin gene in large clostridial glucosylating toxins (LCGTs) secretion, we focused on the role of TcsE in the secretion of TcsL
The are best-understood bacterial secretion systems monoderm organisms, such being as Tat and Sec, highly specific, with amino acid signalsinwithin the secreted proteins and

Summary

Introduction

Introduction distributed under the terms andProtein secretion, defined as the transport of proteins from the cell cytoplasm to the extracellular milieu or into a target cell [1], is a critically important process in all bacteria. The secretion process involves proteins crossing the cytoplasmic membrane (CM), transferring through the periplasm, and traversing the outer-membrane [1]. To achieve this complex process, diderm bacteria have developed a number of distinct secretion systems, with nine major types currently defined [3]. In monoderm (Gram-positive) bacterial cells, this process appears to be simpler since proteins must only cross a single cellular membrane to be secreted [1]. Most proteins secreted by monoderms utilise two main pathways to cross the CM: the general secretion (Sec) or the twin arginine translocation (Tat) pathway [4]. Transport by either system requires that the substrate protein contains a specific secretion signal recognition sequence, or signal peptide, at its N-terminus [2]

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Conclusion