Holin-dependent secretion of a large clostridial cytotoxin by C. perfringens

Abstract

The clostridia are gram-positive, spore-forming, anaerobic pathogens capable of causing various diseases in both humans and animals. To do so, clostridial pathogens secrete a number of lethal toxins each associated with specific diseases. One family of proteins secreted by the clostridia are the large clostridial toxins (LCTs). These LCTs include TcdA and TcdB in C. difficile, TcsL and TcsH in P. sordellii, and TpeL in C. perfringens. LCTs bind and inactivate small GTPases in host mammalian cells, shutting down cell-signaling and eventually leading to cell death. Intriguingly, LCTs all lack any conventional signal peptide, rendering them incapable of being translocated by the Sec- or Tat-secretion system. However, according to recent publications, each LCT has been shown to be dependent on a cognate holin located in their pathogenicity loci. The holins for C. difficile and P. sordellii share similar membrane topologies to that of the phage lambda holin. However, TpeE in C. perfringens shares a similar membrane topology to TatA, a key protein in the Twin Arginine Transport (Tat) secretion system. This thesis tests two models, a pore-forming model and a membrane destabilization model that may explain the mechanism behind TpeE-dependent secretion of TpeL in C. perfringens. The pore-forming model was tested using FAST fluorescence technology and cell-surface biotinylation to determine if the amphipathic helix of TpeE changes its orientation in the presence of TpeL. Using FAST fluorescence, the mean pixel intensity for each frame did not change between TpeE-FAST and TpeE-FAST TpeL cultures, using a membrane impermeable dye. This suggested that the amphipathic helix does not flip into the cell-membrane in the presence of the toxin. Unfortunately, the cell-surface biotinylation was unable to support this as it yielded inconclusive results due to unforeseen errors within the experimental design. However, attention shifted to a proposed membrane destabilization model for TatA which places the focus of the mechanism on the length of the transmembrane helix of TpeE. To test this, anti-His6 western blotting is being conducted to observe a difference in secretion efficiency of lengthened and shortened TpeE to the wild type. We believe that this system may utilize a membrane destabilization model that represents a novel and ubiquitous secretion method that shares aspects of both holin-dependent and Tat-secretion systems.