Bacillus anthracis is a facultative extracellular bacterial pathogen that causes cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are known to provide protection of limited duration and may not protect against the respiratory form of the disease. Commercially available live vaccines for animals have been shown to cause disease in certain species. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective CMI and antibody responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce antibodies, the pag gene encoding PA was expressed in B. abortus RB51, producing a dual vaccine to protect against both brucellosis and anthrax. In a previous study, the entire pag gene was expressed in strain RB51 and following immunization the vaccine induced antibodies against PA in A/J mice. However, PA stability and protective efficacy were less than desirable as only 1/6 were protected. The studies in this dissertation involved synthesizing a gene corresponding to domain 4 (PA4) of the pag gene utilizing the native codon usage of Brucella. The PA4 domain was fused to Brucella signal sequences of Brucella 18kDa protein, superoxide dismutase or no signal sequence to localize the PA4 to the outside cell envelope, periplasmic space or cytosol respectively. Comparisons of the expression level and stability of the native and synthetic PA4 in B. abortus strain RB51 were assessed by immunoblot. The protective efficacy of PA4 expressed in Brucella was assessed by immunization and protection studies in A/J mice against a live challenge with either B. abortus or B. anthracis Sterne spores. ELISA and western blot indicate the induction of PA specific antibodies by these recombinant strain RB51 vaccine constructs. Results based on subisotype antibody ELISA (IgG, IgG1, IgG2a and IgM) and CMI assays (cytokine ELISA of IL-4 and INF-g, and LPA) suggest a Th1 based immune response to strain RB51 and PA. B. abortus strain RB51 expressing PA4 fused to the signal sequence of Brucella 18kDa protein was able to induce 50% protection, while strain RB51 expressing PA4 with no signal sequence gave 17% protection against B. anthracis Stern spore challenge. Mice were boosted with an intraperitoneal injection of purified PA after an initial immunization with Brucella vaccine candidates, sterile saline or pure PA. Protection assessed by live challenge with B. anthracis Sterne spores increased following boosting with PA in 4 cases. Immunization with purified PA, and 3 strain RB51/PA vaccines and a PA boost gave protection against a spore challenge ranging from partial to full. This study suggests that additional work is needed to define the antigens of B. anthracis involved in the induction of specific CMI. / Ph. D.

Sporulation is a process of differentiation that allows capable cells to go into a dormant and resistant stage of life. To become active again, the spores must germinate into vegetative cells. One key process in spore germination is hydrolysis of the cortex peptidoglycan. This process has been studied in a variety of sporulating species; however, it has not been studied in <i>Bacillus anthracis</i>. A clear understanding of cortex degradation may provide information that will allow for better cleanup of spore contaminated sites. The structure of cortex peptidoglycan of <i>Bacillus anthracis</i> was characterized. The peptidoglycan of the dormant spores was extracted, digested with Mutanolysin, and analyzed using HPLC to determine the structure. The analyses revealed that the cortex peptidoglycan of <i>B. anthracis</i> was very similar to other <i>Bacillus sp.</i>. Spores were stimulated to germinate and cortex peptidoglycan was extracted and analyzed at various times. <i>Bacillus anthracis</i> appeared to hydrolyze its cortex more rapidly than other <i>Bacillus </i>species. While the spores of three species release the spore solute dipicolinic acid and resume metabolism at similar rates, the <i>B. anthracis </i> spores released 75% their cortex material within 10 minutes while the other species released only 20% in the same time frame. This suggests that the <i>B. anthracis</i> spore coats are more permeable to cortex fragments than those of the other species, or that <i>B. anthracis</i> rapidly cleaves the cortex into smaller fragments. Novel cortex fragments analyzed during <i>B. anthracis</i> germination were produced by a glucosaminidase; however, additional studies need to be performed for confirmation. / Master of Science

Bacillus anthracis has been classified as one of the most dangerous bioterrorism agents causing high mortality rates in short periods of time. Anthrax spores are extremely resistant to chemical and environmental factors, and have the ability to return into a vegetative (virulent) state during the process of germination. Previous research has suggested that spores can be eradicated with common disinfectants after germination and release of spore coats. During germination, the spore coat is degraded, making the spore susceptible to penetration of chemicals into the spore core. While previous research has focused on a qualitative understanding of germination of spores by obtaining high-resolutions images of spore coats to understand how protein coat layers change during germination, very few studies have evaluated changes in mechanical properties of spores during germination, and how germination affects virulence of macrophages. In this study, we performed a series of in vitro experiments to do an in-depth analysis of germination and virulence of B. anthracis. Atomic force microscopy (AFM) was used to investigate changes in spore surface properties during germination including morphology, roughness, elasticity, and spring constant. AFM results suggested that germination mechanisms depend on germinants used to trigger germination and roughness of Bacillus species increase during germination. In addition, the elasticity and spring cell constant of B. anthracis spores are affected during germination since the elastic moduli and cell spring constant values decreased with time as the spore was germinating, making the cells more susceptible. Spore killing was also tested both in sporulated and vegetative B. anthracis using the antimicrobial peptide chrysophsin-3 and the surfactant dodecylamine (DDA). Both killing agents were capable of eradicating B. anthracis spores, but more killing was observed for spores that were germinating or had become vegetative. The presence of germinant receptors from the Ger operon and its role on germination kinetics of B. anthracis was also investigated. The germination of mutant spores that carried one receptor or lacked all germinant receptors was compared to the germination kinetics of wild-type B. anthracis. Our results suggest that germination of spores is modified by the presence or absence of germinant receptors. Furthermore, the mutant B. anthracis strain lacking all receptors germinated suggesting that other receptor independent pathways may exist in B. anthracis. Finally the ability of B. anthracis to adhere, grow, and invade macrophages was investigated. Invasion of macrophages by B. anthracis was dependent on germinant receptors and the ability of spores to germinate and multiply. Our results suggest that macrophages were not capable of killing infecting spores, and on the contrary, germination of spores inside macrophages caused the lysis of macrophages. An uncontrolled release of cytokines by macrophages was elicited by spores and germinated B. anthracis. Our study helps understand the process of germination of B. anthracis spores at a nanomolecular level. Our investigation may be a valuable tool in the design and development of antisporal compounds.

Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding. / Bakterielle Protein-Toxine gehören zu den wirksamsten bekannten Toxinen. In vielfältigen Variationen findet man sie in allen Bereichen des Lebens. Einige werden von den Bakterien während einer Infektion freigesetzt und übernehmen einen wichtigen Part in der Pathogenität. Andere werden zu Verteidigungszwecken sekretiert und können in verdorbenen Lebensmitteln gefunden werden. Was die Wirkung binärer Toxine der AB7-Gruppe angeht, so gehören diese zu den potentesten und gefährlichsten Giften weltweit. Selbst kleine Mengen dieser Proteine können schwerste Symptome während einer Infektion mit Bakterien der Gattung Clostridium oder Bacillus verursachen. Abgesehen von der Bedrohung die durch die Toxine ausgeht, zeichnen sie sich durch einen einzigartigen Intoxikationsmechanismus aus. AB7-Toxine sind aus einer porenformenden Domäne B, die als eine Art molekulare Injektionskanüle fungiert, und enzymatisch aktiven Proteinen A zusammengesetzt. Der komplexe Wirkmechanismus ermöglicht es nicht nur Zellen in höchst effektiver Weise abzutöten und sollte deswegen zu Behandlungszwecken untersucht werden, sondern könnte auch als molekulares Werkzeug umfunktioniert werden, um spezielle Zellen mit gewünschten Proteinen zu beladen. Für beide Zwecke (Blockierung und gezielter Transport) ist die Bindung an, und der Transport durch die porenformende Domäne von größter Bedeutung. Die Struktur der B-Domäne ist wichtig um tiefere Einsicht in den Transportprozess der binären Toxine zu ermöglichen. Leider ist es bisher nicht gelungen die Kristallstruktur des Membranproteins protective antigen (PA) von Anthrax oder irgendeiner anderen Bindedomäne eines AB7-Toxins zu lösen. Deshalb wurde in dieser Arbeit ein elektrophysiologischer Ansatz zur Bestimmung der biophysikalischen Konstanten des Prozesses gewählt, die Black-lipid-Bilayer Methode. Zusätzliche Versuche an Zellen und mit Mutanten der Proteine dienen zur Absicherung der in vitro Ergebnisse. Im ersten Teil der Arbeit wird der Translokationsmechanismus, und die mögliche Nutzung dessen als molekulares Werkzeug näher behandelt. Dies erfolgte durch Bindungsstudien an PA und C2II mit Effektoren (sowohl aus der AB7-Familie, als auch nicht näher verwandt). In Kapitel 2 wird beschrieben, dass ein His-Tag es EDIN (von S. aureus) und anderen Effektoren ermöglicht, dass ein Transport durch PA-Poren in vitro und in vivo stattfindet (nicht aber durch C2II). Ebenfalls konnte eine starke Abweichung in der bekannten Spannungsabhängigkeit von PA festgestellt werden, die den Prozess eindeutig mit den Membranpotentialen in Verbindung bringt, die häufig eine wichtige Rolle im Metabolismus spielen. Kapitel 3 fasst zusammen, dass Bindungspartner von PA bestimmte Motive beinhalten, die von Substraten wie Ionen in Elektrolyten, über kleine Moleküle, bis hin zu komplexen Proteinen reichen. Diese Erkenntnisse könnten genutzt werden um Blockersubstanzen zur Behandlung von Anthrax, oder Tags zur Aufnahme durch Anthrax zu designen. Neueste Befunde zu Homologien und Unterschieden zwischen den Komponenten der binären Toxine sind der Kern von Kapitel 4, in dem die Kreuzreaktivität von Anthrax und C2-Toxin analysiert wurde. Die enthaltenen Daten ermöglichen einen tieferen Einblick in die verschiedenen Stufen der Bindung und Translokation des edema factor (EF), des lethal factor (LF) und von C2I an und durch PA und C2II. Im zweiten Teil rückt die Blockierung der Intoxikation in den Fokus. Die Analyse speziell designter Blockersubstanzen für PA wird in Kapitel 5 behandelt. Diese formen einen Porenverschluss, der weiteren Transport von Toxinkomponenten verhindert. Eine besondere Bedeutung könnten diese Substanzen im Zusammenhang mit Multiresitenz bei Anthrax Stämmen (vermutlich in Russland als biologische Waffe hergestellt) zur Verhinderung von Symptomen und der Verlängerung der Zeit spielen, die man hat um neue Antibiotika zu erzeugen. Kapitel 6 beschreibt zum ersten Mal die Blockierung von PA-Poren mittels eines Anti-His Antikörpers von der trans-Seite aus. Interessanterweise trat diese Ionenstärke abhängige Blockierung, auch bei einer Histidin zu Glycin Mutation an der Stelle 310 (vermutete Bindeposition) auf.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 201-231).

Thesis (Ph.D.)--George Mason University, 2009. / Vita: p. 102. Thesis director: Serguei G. Popov. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Title from PDF t.p. (viewed June 10, 2009). Includes bibliographical references (p. 84-101). Also issued in print.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Inaug.-Diss.)--Universität Basel, 1896? / Bibliography: p. 33-34.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 2002. / Includes bibliographical references. Also available on the Internet.

Thèse (Ph. D.)--Université Laval, 2008. / Bibliogr.: f. 174-206. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.