Clostridia 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. / Doctor of Philosophy / Clostridium perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals worldwide. C. perfringens is an important organism to study due to its roles as a decomposer in our ecosystem and its ability to cause a number of diseases. These diseases cause considerable harm to livestock and poultry industries, as well as to human and animal life. Though these diseases vary wildly, they share this in common: they are defined by specific toxins and what defines a harmful lineage of C. perfringens is its ability to produce and secrete these toxins. In fact, this is the common denominator to all clostridial diseases, including the notorious diseases C. difficile-associated colitis, tetanus, botulism, and gas gangrene. Of primary concern in diseases caused by C. perfringens and other clostridia is that effective, novel therapies are grossly lacking. The effects of these powerful toxins can outpace antibiotic therapy and this often leads to extended periods of suffering, even in favorable cases. Of all the pathogenic clostridia, C. perfringens is easy to test in the laboratory and may even be used in place of more dangerous and difficult to work with bacteria. This is useful in developing better treatments and for studying treatment applications for the toxins themselves! Indeed, bacterial toxins have beneficial applications, Botox being a good example, as well as in cancer treatments.
Like many other bacteria, C. perfringens can produce strong, rope-like appendages called pili that are made and extended and retracted from the cell, similar to a lasso, by the concerted effort of many different proteins. These pili confer a number of advantages for bacteria, one being a means for attachment to host cells, an important first step in establishing an infection. With the overarching goal toward sowing future therapeutic developments, Chapter 2 describes an experimental plan to identify and understand the genes for the proteins that allow C. perfringens to attach to muscle cells. Preliminary results are presented for a proof-of-concept method, which was ultimately discontinued November 2016 because reliable, robust results were not obtained.
In addition to host cell attachment, pili have been shown to function a sensor for cell. For example, in another bacterium, pili "sense" a suitable surface for attachment and interpret this signal so the bacterium can attach and "set up shop" by releasing toxins. Based on considerable evidence, we thought C. perfringens might also "sense" a surface with its pili and interpret this signal for attachment and cell growth by means of interactions between three specific proteins. We designed a series of experiments to test this hypothesis, but due to the failure of important, initial studies, this project was discontinued in November 2017.
Even though all clostridial diseases are caused by toxins, which must be secreted outside the bacterium to do harm, how these toxins are secreted is poorly understood. In Chapter 4, we investigate a toxin secretion method where a certain type of toxin is thought to be secreted through a temporary hole formed by many copies of a small, partner. First, we forced C. perfringens bacteria to artificially produce both the small protein and the toxin and found that the toxin needs this small protein to be secreted. We then deleted parts of both the toxin and the small protein and determined which parts of each are essential for this secretion method by linking an absence of secretion in bacteria whose proteins are missing essential parts. Further, we determined that production of this small, partner protein was kept to low levels and was harmless in C. perfringens, but was lethal in a different, unrelated bacterium, Escherichia coli, implying that C. perfringens bacteria have an ability control this hypothetical hole in themselves that E. coli does not. Our results, in conjunction with those from previous groups, suggest a pattern for secretion of this type using these small proteins. This information is a key first step towards developing better therapies for clostridial diseases, since without toxin secretion, clostridial diseases cannot occur.
Chapter 5 describes the surprising outcome of an experimental approach where the toxin and small partner protein are produced in the bacterium by two different mechanisms. We found a number of off-target effects associated with this approach, one of which was the strange facilitation of off-target protein secretion. These off-target effects confused our experimental results and since it was likely that future experiments would also be uninterpretable, we abandoned this approach and used a simpler one instead.
The mechanisms for toxin (TpeL) secretion by its small, partner protein (TpeE) are unclear. A key, initial step towards understanding this mechanism is to determine if the two proteins are in close enough proximity to one another in the bacterium. We developed a strategy to determine if the proteins are close enough together in the cell that takes advantage of the considerable size difference between the two proteins. Presented in this chapter are several initial experiments that can enable this experiment in the future.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/111126 |
| Date | 11 January 2021 |
| Creators | Saadat, Angela P. |
| Contributors | Graduate School, Melville, Stephen B., Stevens, Ann M., Young, Ryland, Hauf, Silke |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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