ABSTRACT: Large clostridial toxins (LCTs) are secreted virulence factors found in several species, including Clostridioides difficile, Clostridium perfringens, Paeniclostridium sordellii, and Clostridium novyi LCTs are large toxins that lack a secretion signal sequence, and studies by others have shown that the LCTs of C. difficile, TcdA and TcdB, require a holin-like protein, TcdE, for secretion. The TcdE gene is located on the pathogenicity locus (PaLoc) of C. difficile, and holin-encoding genes are also present in the LCT-encoded PaLocs from P. sordellii and C. perfringens However, the holin (TpeE) associated with the C. perfringens LCT TpeL has no homology and a different membrane topology than TcdE. In addition, TpeE has a membrane topology identical to that of the TatA protein, which is the core of the twin-arginine translocation (Tat) secretion system. To determine if TpeE was necessary and sufficient to secrete TpeL, the genes from a type C strain of C. perfringens were expressed in a type A strain of C. perfringens, HN13, and secretion was measured using Western blot methods. We found that TpeE was required for TpeL secretion and that secretion was not due to cell lysis. Mutant forms of TpeE lacking an amphipathic helix and a charged C-terminal domain failed to secrete TpeL, and mutations that deleted conserved LCT domains in TpeL indicated that only the full-length protein could be secreted. In summary, we have identified a novel family of holin-like proteins that can function, in some cases, as a system of protein secretion for proteins that need to fold in the cytoplasm.IMPORTANCE Little is known about the mechanism by which LCTs are secreted. Since LCTs are major virulence factors in clostridial pathogens, we wanted to define the mechanism by which an LCT in C. perfringens, TpeL, is secreted by a protein (TpeE) lacking homology to previously described secretion-associated holins. We discovered that TpeE is a member of a widely dispersed class of holin proteins, and TpeE is necessary for the secretion of TpeL. TpeE bears a high degree of similarity in membrane topology to TatA proteins, which form the pore through which Tat secretion substrates pass through the cytoplasmic membrane. Thus, the TpeE-TpeL secretion system may be a model for understanding not only holin-dependent secretion but also how TatA proteins function in the secretion process.
