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.

Staphylococcus aureus is a gram positive bacterium asymptomatically colonizing 30-40% of the human population. S. aureus causes a variety of infections including superficial skin lesions, toxic shock syndrome, and infective endocarditis (IE). There are 100,000 cases of IE each year in the United States. IE is a life threatening infection of native/prosthetic valves and the lining of the heart. It is characterized by the formation of vegetations, “cauliflower-like” structures composed of bacteria and host factors. S. aureus is the most commonly identified pathogen (up to 40%) in patients with IE. USA200 (Clonal Complex 30) strains of S. aureus are significantly associated with IE, all of which produce toxic shock syndrome toxin-1 (TSST-1) and β-toxin. TSST-1 characterizes the staphylococcal Group I superantigens (SAgs). The major mechanism of activity of TSST-1 and other SAgs is the ability to activate T-cells and APCs by non-specifically cross-bridging Vβ-chains of T-cell receptors (TCRs) with α and/or β-chains of major histocompatibility complex II (MHCII) molecules on antigen presenting cells (APCs). In a rabbit model of IE and sepsis, TSST-1 is critical for the development of vegetations and the associated colony forming units (CFUs). β-toxin has a molecular mass of 35 kDa, a basic pI (>10.0), and is a member of the DNase I superfamily. This cytotoxin has two distinct mechanisms of action: sphingomyelinase (SMase) activity and DNA biofilm ligase activity. β-toxin is critical for causing IE in a rabbit model that strongly resembles human disease. This toxin association had been observed, but studies have not been completed to determine what role TSST-1 and β-toxin play independently and in cooperation with one another, and more specifically which mechanism each uses, during IE infections. While TSST-1 and β-toxin are both important for IE, they are very different toxins. My studies determined that the presence of TSST-1 and β-toxin in combination results in the highest levels of lethality in a rabbit model of IE. A strain expressing TSST-1 lacking superantigenic activity has decreased lethality compared to the same strain expressing wild type TSST-1. My study is the first to begin characterization of the DNA biofilm ligase active site by identifying important residues via a DNA binding and biofilm formation assays. Furthermore, my research shows that a β-toxin mutant lacking SMase activity is decreased in lethality and vegetation formation compared to wild type. β-toxin mutants disrupted in biofilm ligase activity do not decrease lethality but are deficient in vegetation formation compared to wild type. Utilizing in vitro assays to assess cellular events during IE, I established that β-toxin causes changes to morphology and is cytotoxic to human aortic endothelial cells (HAECs), inhibits production of IL-8, and modulates the expression levels of cluster of differentiation 40 (CD40) and vascular cell adhesion molecule 1 (VCAM-1). My work shows these two virulence factors (TSST-1 and β-toxin) produced by USA200 strains and other clonal groups play important roles in causing IE.

This work is focused on the RTX (Repeats in ToXin) domains structure of selected RTX toxins and its impact on secretion and protein folding. The structural analysis included RTX domains of ApxI (Actinobacillus pleuropneumoniae-RTX-toxin I) from Actinobacillus pleuropneumoniae, HlyA (Alfa-hemolysin) from Escherichia coli and LtxA (Leukotoxin A) from Aggregatibacter actinomycetemcomitans and blocs 4 a 5 RTX domain CyaA (adenylate cyclase toxin) from Bordetella pertussis. The structures of LtxA RTX domain and CyaA RTX blocs 4 and 5 were obtained and characterized. Two models of CyaA RTX domain were built based on SAXS (Small Angle X-ray Scattering) model, previously solved RTX structures and RTX structures presented here.

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Bacillus subtilis is a gram positive soil bacterium that is surrounded by many other microorganisms its environment. That is why it is necessary for the bacterium to be able to fight with these microorganisms for the nutrients and living space. B. subtilis contains the modules in its genetic make-up that improve its ability to compete. These modules are called the toxin-antitoxin systems. This Diploma Thesis is trying to identify yet undescribed extracellular toxins produced by the wild type BSB1 strain of B. subtilis. The related microorganism Bacillus megaterium was used as a competing bacterium. The contact-dependent or independent manner of killing the competing bacterium was demonstrated using this model. By deletion analysis and comparisons of the genomes of the various strains of B. subtilis, the SPβ prophage was first identified as a region containing an unknown toxin(s). Analysis of the extracellular proteome of B. subtilis subsequently revealed an unknown toxin (or toxin complex, respectively) of the molecular weight exceeding 100 kDa. Even more fascinating was the finding that such a large protein molecule is resistant to the pancreatic protease, trypsin. Subsequent non-enzymatic cyanogen bromide cleavage of the extracellular proteins and their analysis by mass spectrometry revealed...

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Bordetella pertussis produces a number of virulence determinants believed to contribute to its survival in the host as well as to the pathogenesis of disease. One of these factors, adenylate cyclase toxin (ACT), has been implicated to penetrate human neutrophils and macrophages and abrogate their function by virtue of unregulated production of intracellular cAMP. In order to adequately study the nature of ACT and its role in pathogenesis, it is necessary to isolate the toxin from other virulence factors produced by the organism. Attempts by other investigators to purify ACT and maintain both its invasive and catalytic properties have not been successful. B. pertussis produces a cell associated ACT during mid-log phase of growth in Stainer-Scholte medium. Purification of ACT with both activities from urea extracted whole cells has been achieved by hydroxylapatite and calmodulin-sepharose chromatography. ACT is a single protein of 220 kd molecular weight with an isoelectric point of 7.0. The protein probably contains regions which are strongly hydrophobic. ACT has a specific activity of nearly 17,000 μM cAMP formed/min. An 850 ng sample of ACT induced over 1,400 pmoles cAMP/10⁶ S49 mouse lymphoma cells while 660 ng of ACT inhibited human neutrophil chemiluminescence by 65%.

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