Saadat, Angela P.2022-07-062022-07-062021-01-11vt_gsexam:28585http://hdl.handle.net/10919/111126Clostridia are a class of gram-positive, anaerobic bacteria best known for their powerful toxins. These bacteria cause many diseases that are difficult to treat and often deadly, including colitis, botulism, tetanus and gas gangrene. These diseases are caused by the secretion of specific toxins, though current treatments do little to nullify these toxins and better therapeutics are urgently needed. The development of such treatments is hindered by our poor understanding of clostridial toxin secretion, which is itself hindered by the innate characteristics of these bacteria that make them difficult to study. Of the pathogenic clostridia, Clostridium perfringens is relatively easy to culture and straddles the line between pathogen and commensal, making it an attractive model organism for studying clostridial toxin secretion. C. perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals that can also cause disease. C. perfringens produces more toxins than any other bacterium, and these toxins generally function as a means to lyse host cells so the bacteria may scavenge their intracellular nutrients. The primary focus of the research in this dissertation is the secretion of the toxin TpeL by a small membrane protein, TpeE. Preceding the study of TpeL secretion were two other projects, which are discussed in Chapters 2 and 3. Chapter 2 describes an experimental plan to characterize the genes involved in muscle cell adherence as a very basic model to mimic skeletal muscle attachment in gas gangrene. Like many other bacteria, C. perfringens can produce T4P, extracellular filaments that are synthesized, extended and retracted from the cell by the concerted effort of many proteins. Results from initial, proof-of-concept adherence assays are presented and demonstrate that statistical significance was lost when data were compiled. Despite efforts to troubleshoot this, robust test output was not achieved and the project was discontinued November 2016. Chapter 3 describes the experimental plan and initial findings of a project where a link between T4P and virulence was investigated. Such a link had been demonstrated in the T4P model organism Pseudomonas aeruginosa, where PilT, the T4P retraction ATPase, was shown to sense surface attachment and initiate virulence. In C. perfringens, PilT demonstrates a number of characteristics that lead us to think it may also function as a sensor, coordinating host cell attachment and colonization by alternatively associating with PilM and FtsA. We developed an experimental plan to determine if PilT binds both PilM and FtsA by co-immunoprecipitation with live-cell fluorescence imaging. However, we were unable to demonstrate the functionality of a PilT-fluorescent protein fusion with an anti-pilin ELISA assay, nor were we able to detect PilT or FtsA overexpression by immunoblotting, and the project was discontinued in November 2017. In retrospect, these experiments likely failed because of an inactive promoter region in the overexpression plasmid. Though clostridial diseases require secreted toxins, their secretion mechanisms are largely uncharacterized, and Chapter 4 describes the investigation of a potentially conserved toxin secretion mechanism. TpeL is a recently discovered C. perfringens toxin that is associated with chicken necrotic enteritis, a disease that costs the poultry industry billions of dollars each year. TpeL belongs to a subset of clostridial toxins characterized by their large size and conserved structure, the large clostridial toxins. The gene for tpeL and nearly all other large clostridial toxins lies next to a gene encoding a small membrane protein. Since bacterial genes with a shared function are often found in close proximity, it is suspected that these small proteins share some function with these toxins, and another research group has shown the two large clostridial toxins in C. difficile need this small membrane protein for their secretion. We isolated the small membrane protein and toxin genes tpeE and tpeL from native regulatory elements and overexpressed them heterologously in a different strain of C. perfringens. By immunoblotting, we found rapid TpeL secretion requires TpeE, and secretion was abolished when C-terminal sections of either protein were mutated. By immunoblotting and growth curve analyses, we found that TpeE is maintained at low concentrations and is not lethal in C. perfringens, but was expressed to high levels and was lethal in Escherichia coli. Our results, in conjunction with those from other research groups strongly suggest a conserved secretion mechanism dependent on small, membrane proteins. Our findings further the understanding of toxin secretion, a key step toward novel and effective clostridial disease strategies. Chapter 5 describes the outcome of an experimental approach where tpeE and tpeL were expressed from two different expression system plasmids. A number of off-target effects materialized with this approach which confounded our experimental results. The predominantly confounding effect was off-target protein secretion, found by immunoblotting to be associated with one of the expression systems. Despite efforts to minimize these effects, it became clear results from this approach would be uninterpretable and the two-plasmid approach for TpeE and TpeL expression was abandoned. A cut-and-paste strategy using the historical, single inducible expression system was implemented in its place. The exact mechanism for TpeL secretion by the small membrane protein TpeE is unclear. Chapter 6 outlines some hypotheses towards this mechanism and a nascent plan to uncover it. An efficient starting point is to determine if the two proteins are in close enough proximity to one another to interact in vivo. We developed a strategy to determine this by crosslinking and immunoblotting, using the size differential between the proteins to our advantage. Though the results of this study were confounded by an inability of TpeL to solubilize in buffer, the groundwork is laid for future endeavors.ETDIn CopyrightbacteriaclostridiapathogentoxinsecretionDissertation