Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. The two principle virulence factors, TcsH and TcsL, are highly homologous to C. difficile TcdA and TcdB, respectively. Experiments to compare the effects of TcsH and TcsL to TcdA and TcdB show that TcsH induces necrotic cell death similarly to TcdB while TcsL induces apoptotic cell death. Further work focuses on TcsL, which has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. The use of conditionally immortalized murine pulmonary microvascular endothelial cells provides a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities are introduced into a recombinant version of TcsL, and the activities of these mutants are compared to those of wild-type toxin. All mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL.

Obesity is a serious health issue in the United States as well as worldwide and contributes to several other disorders such as cardiovascular diseases, type II diabetes and asthma. However, the impact of obesity on immune responses to infections is poorly understood. During the last decade, specifically after the 2009 H1N1 strain influenza pandemic, evidence has emerged that obesity increases the risk and worsens the outcome for infection by influenza A virus (IAV). Obese individuals were hospitalized at a higher rate compared to their lean counterparts, had longer hospitalization times, responded poorly to vaccination and had increased mortality. Host protection and clearance of respiratory pathogens like IAV require robust pulmonary immune responses. Since alveolar macrophages (AM) are the first line of defense against respiratory pathogens, we hypothesized that obesity-associated alterations in AM functions worsen IAV infection outcomes. To test this hypothesis I fed C57BL/6 mice with a low-fat or high-fat diet for different time periods and infected these animals with an H1N1 laboratory strain of IAV (PR/8). Mice on the high-fat diet (obese mice) showed increased morbidity and mortality upon infection as compared with mice on the low-fat diet (lean mice). I also found that obesity induces changes in overall lung cellularity, AM polarization and phagocytic ability, and alterations in the level of surface proteins such as Gal-3, MHC-II, Tim-3, and cytokines such as IL-6, TNF-?, IFN-? and IL-10 in the lung. These obesity-induced changes in AM may contribute to impaired immune responses during IAV infection, resulting in worsened outcomes.

Achieving transplant tolerance in the autoimmune environment will require targeting multiple immunologic dysregulations in T-B lymphocyte collaboration that drive the aggressive anti-graft response. At a biologic level, my findings reveal the necessity of overcoming B lymphocyte mediated restriction of CD4 Treg function, failed HSC mobilization, and enhanced T cell metabolism in achieving transplant tolerance in murine models of Type 1 Diabetes (T1D) and Systemic Lupus Erythematosus (SLE). At a cellular level, my dissertation demonstrates that specific failures in CD4 and CD8 Treg mediated suppression of the effector cell response in autoimmune T1D and SLE contributes to a generalized resistance to transplant tolerance observed in these strains. Overall, identification of and surmounting the key dysregulations in T-B cell collaboration that permit loss of tolerance in autoimmunity will advance the clinical potential of transplantation as a cure for autoimmune disease.

Seasonal influenza remains a worldwide health concern and recently, the novel avian influenza virus, H5N1, has infected and caused disease in humans, though the virus is not currently capable of human-to-human transmission. Since 2003, 850 human cases of the novel influenza virus H5N1 have been reported with a 50% mortality rate. Recently two labs have shown, very few mutations may be necessary for efficient transmission between humans. In order to examine the immune response to H5N1, a panel of antibodies from subjects in a phase I clinical trial of an experimental H5N1 vaccine were isolated and characterized. We choose a potent and specific anti-H5N1 antibody, H5.3, for further studies in order to determine the molecular mechanism of neutralization used by H5.3 and how the antibody developed. The structure of the H5.3 Fab in complex with the H5 head domain showed H5.3 interacts with the highly conserved receptor binding site and polymorphic residues on the edges of the interface, indicating breadth and potency of the antibody conflict due to variability outside the receptor-binding site. As evidenced by the structures of the H5.3 Fab in complex with H5 respiratory droplet transmissible variants, the receptor preference of the virus may not be critically important for recognition by a receptor binding site directed antibody. The H5N1 vaccine elicited a primarily naïve antibody response, as the H5-specific antibodies had a lower number of somatic mutations than the broadly neutralizing influenza antibodies. The H5.3 somatic mutations do not stabilize the protein conformation, as it remains flexibility after affinity maturation, and do not have a large effect on increasing the affinity of H5.3 for H5. Overall, this research will contribute to influenza vaccine design.

My project has focused on understanding the structural organization of the clostridial neurotoxins, botulinum and tetanus, that are the causative agents of the neuroparalytic diseases botulism and tetanus. Once the toxins enter the bloodstream they inhibit the release of neurotransmitters required for the contraction or relaxation of muscles, resulting in debilitating paralysis. Botulinum neurotoxin is also orally toxic, whereas tetanus neurotoxin is not. Botulinum interacts with additional Clostridium botulinum proteins to form complexes that protect the neurotoxin from degradation as it passes through the hosts gastrointestinal tract. Using electron microscopy techniques, I have produced novel structural insights into the molecular organization of the three botulinum neurotoxin progenitor complexes responsible for human botulism. This dissertation also reports analysis of the tetanus holotoxin structure.

The fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis and has played a role in at least 90 amphibian extinctions since 1980. However, some amphibian species are more resistant to Bd than others, which may be due to one or more innate defenses in the skin, including antimicrobial peptides (AMPs) and symbiotic bacteria that produce antifungal metabolites. In this dissertation, I explored the contributions of these defenses to resistance against Bd in southern leopard frogs, Rana sphenocephala, which required developing methods to reduce or eliminate these defenses in laboratory animals. I examined the ability of skin bacteria to defend R. sphenocephala prior to the development of a substantial AMP defense and identified new symbiotic bacteria capable of inhibiting or enhancing Bd growth in vitro. I more fully characterized a known peptide, Peptide C, in R. sphenocephala, quantified the inhibitory activity of four known R. sphenocephala AMPs in vitro, elucidated the AMP defense ontogeny in this species, and determined the impact of induced AMPs on viability of skin bacteria. For some endangered species, captive colonies are the best intermediate solution for long-term survival, and effective antifungal treatments are needed to treat chytridiomycosis and limit Bd spread in captive colonies. Unfortunately, the only currently accepted treatment uses itraconazole, which can be toxic. In studying alternative anti-chytrid drugs, I quantified the minimum inhibitory concentrations of nikkomycin Z, chloramphenicol, and amphotericin B and undertook the first analysis of the interactions and effects of these drugs on amphibian innate immune defenses. In total, my studies shed light on two important immune defenses against Bd and contribute to the search for new drugs against a significant fungal pathogen endangering amphibians worldwide.

Allergic asthma is refractory to corticosteroid treatment in up to 10% of patients and often leads to hospital admissions caused by respiratory viral and/ or bacterial infections. In these studies, I found that: 1) STAT6 inhibited innate γδ17 cell immune responses. STAT6 suppression of γδ17 cell function may provide one explanation for why asthmatic patients have significantly greater risk for invasive bacterial disease, including pneumonia, than nonasthmatic subjects. 2) A GLP-1R agonist, an FDA-approved agent currently used for Type II Diabetes, attenuated the type 2 immune response to RSV and attenuates RSV illness. The current availability of GLP-1R agonists for human treatment highlights the clinical significance of these studies as this therapy could be immediately transferrable to RSV disease. 3) PGI2, an FDA-approved agent currently used for pulmonary hypertension, protected against autoimmunity; enhanced Treg stability and function; rendered T effector cells more susceptible to Treg- mediated suppression; and promoted iTreg differentiation. PGI2 may therefore represent a novel treatment strategy for diseases that result from Treg dysregulation.

This thesis advances our understanding of circulating B and pTFH cells in untreated chronic HIV+ infection. My studies determined the frequency, phenotype, and functional response to antigen stimulation of pTFH and B cells in chronically infected HIV+ individuals. I found that while B cells were activated at baseline and had high PD-1 expression, antigen responses were improved with CD4+ T cell help and PD-1 blockade. pTFH cells, the best providers of B cell help, had decreased maximal responses to stimulation as measured by expression of CD40L and ICOS, but maintained responses to recall antigens. The fact that decreased maximal responses did not correlate with any clinical aspects of disease could suggest that pTFH cell dysfunction may occur early in HIV infection. Functional pTFH cell responses to recall antigen existed in HIV+ individuals but also did not correlate with clinical aspects of disease. This maintenance of HIV-specific pTFH cells may simply be a result of HIV viremia driving chronic immune activation. However, the existence of recall tetanus responses suggests that vaccines that elicit protective antibodies may still be effective in HIV+ individuals. Thus, for vaccines designed to elicit antibody responses, adjuvants to optimize TFH cell help may be advantageous. Additionally, pTFH cells should be investigated for a potential role identifying immune correlates of vaccination. As a final component of my thesis, I compared a new technology, mass cytometry, to the current standard technology in the field, fluorescence cytometry, and found they were highly comparable. My results demonstrated that multidimensional analysis combined with flow cytometry will be particularly useful for the comprehensive characterization of cells, including cells with dynamic or unexpected cell phenotypes in health and disease.

The immune system is charged with the task of protecting the body from pathogens and clearing apoptotic or cancerous cells to prevent chronic disease. Controlled cell migration shapes lymphocyte subset development, homeostasis, and immune responses in a lineage specific manner. Although frequently associated with lymphocyte survival and quiescence, one of the primary functions of the transcription factor Krüppel-like factor 2 (KLF2) is to direct lymphocyte migration. In this thesis I report that KLF2 differentially regulates CD4+CD25- and CD8+ T cell migration, and is necessary for natural killer (NK) cell homeostasis. Surprisingly, in CD4+CD25- and CD8+ T cell lineages, KLF2-directed migration patterns are dispensable for effector function but may promote peripheral tolerance. NK cells have been long thought to promote viral clearance and tumor surveillance. In the NK cell compartment KLF2 controls homeostasis by (a) suppressing immature NK cell proliferation and (b) promoting localization toward IL-15 rich niches necessary for mature NK cell differentiation and survival. Through these novel KLF2-directed mechanisms it may be possible to increase the number and persistence of mature NK cells to improve NK cell based cancer therapies.

Bacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this thesis, I describe the isolation and analysis of a membrane-spanning core complex from the <i>Helicobacter pylori cag </i>T4SS, which has an important role in the pathogenesis of gastric cancer. This complex contains five <i>H. pylori </i>proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to<i> H. pylori</i>. Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by<i> Îcag3 or ÎcagT</i> mutants retained the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirmed that Cag3 is a peripheral component of the complex. The <i>cag</i> T4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the <i>H. pylori cag</i> T4SS that are optimized for function in the human gastric mucosal environment.