This work explores bacterial motility from the mechanisms of propulsion of an individual cell to the complex behavior of collective motility. The shear modulus of bacterial flagella was measured by stretching isolated flagella with an optical trap and by measuring force extension curves of the stretched flagella shedding light onto the me-chanics involved in the motility of single micro-organisms. Experiments in concentrated suspensions of bacteria show collective behavior with large scale mixing on a time and length scale greater than can be understood from the standard model of "run and tumble" motility of a single organism are reported. To further understand the transition from individual to collective motility a novel form of motility where an individual bacterium can reverse direction without changing cell orientation is reported here. These experiments further the understanding of bacterial motility.

Borrelia hermsii, an etiologic agent of tick-borne relapsing fever, binds negative complement regulator factor H (FH) via its FhbA protein. Direct demonstration of the role of FhbA in the disease process has been hindered by the lack of genetic manipulation systems for the relapsing fever Borrelia. Here, we demonstrate successful generation of a B. hermsii strain YOR fhbA deletion mutant (Bh YORΔfhbA) that constitutively produces green fluorescent protein (GFP). Genetic manipulation did not affect growth rate or plasmid composition. Bh YORΔfhbA lost factor H-binding and C3b-inactivation capabilities, but retained resistance to killing in human serum and infectivity in mice. Stable production of GFP was demonstrated in vitro and in vivo. Collectively, these results suggest that B. hermsii employs an unidentified mechanism of complement evasion that is FH-independent and sufficient for persistence within the host. Additionally, this study represents a significant methodological advancement in the molecular characterization of relapsing fever spirochetes.

The study of leptospiral pathogenesis is hampered by the lack of efficient mutagenesis methodologies. Thus research has focused on alternative approaches including genome sequencing, comparative genomics, transcriptomics and proteomics. In this thesis a comparative proteomic approach was used to identify leptospiral proteins with a potential role in the leptospiral infection process. Identification of proteins was followed by characterization of target proteins with potential roles in the infection process and ultimately led to the identification of a novel leptospiral virulence factor. Specifically, comparative proteomics using isobaric tags for relative and absolute quantitation complemented with two-dimensional gel electrophoresis were used for mass spectrometry-based protein identification and quantitation. These methodologies were utilised to identify and quantitate leptospiral proteins altered in expression in response to growth media limited in iron supply and/or supplemented with serum. These conditions were designed to mimic a subset of variables encountered by the bacteria within the host. These experiments led to the identification of five proteins with potentially novel roles in the leptospiral infection process. One of these proteins was further characterized as a periplasmic catalase, KatE. Using insertion mutagenesis it was demonstrated that KatE enhances extracellular H2O2 resistance and is required for virulence in guinea pigs and hamsters. Proteomic analyses also led to the identification of glutamic acid methylation of a protein that was further characterised to be surface exposed and expressed during leptospiral colonization of hamster liver and kidneys. This was the first description of glutamic acid methylation of a surface exposed protein in Leptospira. / Graduate

Borrelia burgdorferi is the etiological agent of Lyme disease. Not much is known about the susceptibility of this organism to chemical disinfection. Current antimicrobial susceptibility test methods, such as those published by the American Society for Testing and Materials (ASTM), usually require assessment of the number of colony forming units (cfu) of growing organisms on plates following exposure to an agent. For fast-growing organisms, plates are ready for counting 1-2 days post plating, while several weeks may be needed for slower growing organisms. Spirochetes, like B. burgdorferi are difficult to grow on solid media and typically require long incubation periods, sometimes up to several weeks, to generate visible colonies. These issues make B. burgdorferi cfu assessment by plate counting difficult and unreliable. Furthermore, Borrelia have a demonstrated capacity for pleomorphic forms, and can exist in spirochete, round body, or biofilm forms, depending on culture conditions. Plate counts, by nature, do not allow for assessment of morphological form changes. Additionally, the susceptibility of B. burgdorferi pleomorphic forms to chemical disinfectants has not been tested. In this study, we used the SYBR GREEN I/Propidium Iodide (SG I/PI) viability assay to rapidly estimate the percent kill of B. burgdorferi pleomorphic forms to chemical disinfection. Planktonic spirochete populations in 30-second treated samples showed viability percent values of: >95% for Hanks balanced salt solution (HBSS), ~60% for distilled deionized H2O (dd H2O), <5% for ACS 200, and 1% for 1% glutaraldehyde (GTA). Solutions containing 70% ethanol (ETH) and 1% hypochlorite (HC) showed no viable spirochetes following treatment. The percent of live round body cells following different treatments were: >99% for HBSS and <25% for dd H2O. ACS 200, 1% GTA, and 70% ETH treatments resulted in <1% live round body forms, whereas HC showed no live round cell forms. The susceptibility of B. burgdorferi biofilms to various treatments was also assayed using a SG I/PI viability stain after 30-minute contact times. The percent of viable organisms (green) in the treated biofilms was estimated by microscopic observations. HBSS controls showed >98% of bacteria in the biofilm were alive, while treated biofilms showed the following percent viabilities: ACS 200 - ~2%, 1% HC - <1%, 5% HC - <1%, 1% GTA - ~10%, 70% ETH - ~ 2%, and dd H2O ~40%. These techniques merged standardized assessment of antimicrobial activity in liquid culture using an ASTM-type kill-time procedure with viability techniques used in antibiotic susceptibility testing to rapidly evaluate the percent kill of B. burgdorferi pleomorphic forms in vitro following disinfectant exposure. These results showed that B. burgdorferi biofilm forms are orders of magnitude more resistant to chemical disinfection than other morphological forms of this organism.

Periodontal disease is a progressive inflammatory condition that is characterized by the reabsorption of alveolar bone, the destruction of connective tissue, and edentulism. It is caused by a dysbiosis in the oral microbiome as a result of a shift from a Gram-positive aerobic bacterial population, to one that becomes more Gram-negative and anaerobic. Treponema denticola is thought to drive this diseased state based on its role as a keystone periopathogen. A major component of T.denticola’s invasiveness is its motility, which allows the spirochete to penetrate and disseminate through tissues. This motility, which has been seen to be crucial to the invasiveness in other spirochetes through deletion studies, is often regulated by the second messenger, c-di-GMP. In this study, biochemical and biophysical assays were utilized to determine that the predicted diguanylate cyclase TDE0125 converts GTP to c-di-GMP. This elucidates further function of the c-di-GMP regulatory network in T. denticola.

The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires periplasmic localization. The periplasmic OppAIV-PhoA fusion as well as the cytoplasmic PncA-PhoA fusion produced detectable PhoA protein in E. coli and in B. burgdorferi. The periplasmic fusion construct, but not the cytoplasmic fusion construct, resulted in functional alkaline phosphatase (AP) activity in E. coli, as observed by blue colonies on agar plates containing a chromogenic substrate for AP. In contrast, both of the fusion constructs produced limited detectable levels of functional alkaline phosphatase activity in B. burgdorferi, as observed by yellow color change in liquid protein lysate containing a chromogenic substrate for AP. Development of a PhoA fusion reporter system for use in B. burgdorferi will provide a new molecular genetics tool for analyzing the topology of B. burgdorferi transmembrane proteins. These types of studies are critical for understanding the function of B. burgdorferi transport systems and may identify novel molecular approaches for the treatment of Lyme disease.

This historical case study examined the Federal Theatre's Living Newspaper Spirochete, a dramatized history of syphilis, and its relationship to public health education during the late 1930s. The materials for this historical case study were found in the Library of Congress Federal Theatre Project Special Collection, Fenwick Library, George Mason University, and in materials from the Records of the Works Projects Administration located in the National Archives and Records Administration, Washington, D. C. The study sought to examine and establish Spirochete as an example of a special relationship among government, health, education and the arts. An investigation of SpIrochete's relationship to public health education and an inquiry into Spirochete as a dramatic form provided the basis for the study. The study found that Spirochetes origins lay in the unique socioeconomic and political environment of Chicago, and of the nation, during the mid to late 1930s. Spirochete was similar to other Living Newspapers in its goal to inform audiences about a pressing social problem, to present facts and information, and then to motivate action resulting in social change. Spirochete 's form and techniques incorporated many of those found in other Federal Theatre Living Newspapers including many short scenes separated by Black Outs, experimental staging, and the innovative use of light, sound and spectacle to underscore and forward the dramatic action. Spirochete, however, was unique in its subject, syphilis, in its historical perspective, and its use of dramatized case histories juxtaposed with on-stage demonstrations of medical and scientific progress. The study determined that the more than 100 performances of Spirochete in five cities made significant contributions to health issues and attitudes in the War on Syphilis. Spirochete helped break the silence that surrounded the nation's number one preventable killer and crippler. Spirochete imparted facts about syphilis in dramatic vignettes and with creative, innovative stagecraft. Although Spirochete cannot be considered great theatre, according to the criteria of most theatre authorities, Spirochete was a vibrant, viable form of education. Spirochete was propaganda, presenting a definite idea for a definite purpose. Conclusions drawn from the study indicate that Spirochete was an intentional, unique, and vital weapon in the War on Syphilis. / Ed. D.

This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.

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