No description available.

No description available.

No description available.

Previous human antibody studies have shown that the human VH1-46 antibody variable gene encodes much of the naturally-occurring human B cell response to rotavirus, and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46 encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. Here, I used a hybrid methods approach for antibody epitope mapping including x-ray crystallography, single particle cryo-electron microscopy (cryo-EM), enhanced amide hydrogen/deuterium exchange mass spectroscopy (DXMS) and computational modeling to determine the location and mode of binding of an inhibitory (RV6-26) and non-inhibitory (RV6-25) VH1-46 antibody. These studies showed that the location of binding of the non-inhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of the inhibitory antibody RV6-26 deeper in the transcriptional pore to sterically block the pore and hence inhibit transcription. In addition, these studies reveal intricate binding patterns of both antibodies to VP6 that inform on the structural arrangement of VP6 molecules in the virion particle.

Helicobacter pylori colonizes the human stomach and is associated with an increased risk of peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma. An important virulence factor produced by Helicobacter pylori is the secreted pore-forming toxin known as VacA. The 88 kDa secreted VacA toxin is comprised of two domains, designated p33 and p55. The overarching objective of my thesis work was to analyze the structure and function of the VacA p33 domain. I successfully purified a recombinant form of the p33 domain, showed that it physically interacted with the p55 domain to form complexes, and demonstrated that toxin activity could be reconstituted by mixing the p33 and p55 domains. Electron microscopy of p33/p55 complexes revealed small rod-shaped structures that converted to oligomeric flower-shaped structures in the presence of detergent. I investigated functional properties attributable to the p33 i-region, and showed that polymorphisms in this region were associated with variations in the ability of VacA to cause alterations in human T cells. An increased activity of type i1 forms of VacA compared to type i2 forms of VacA was attributable to differential cell-binding properties of these proteins. Finally, I successfully crystallized an 88 kDa VacA protein secreted by H. pylori and demonstrated that it is comprised of predominantly β-helical elements. Taken together, my work has demonstrated the importance of the p33 domain in VacA activity, and has provided new insights into the structure of the p33 domain.

Global amphibian populations, including the northern leopard frog (Rana pipiens), have been declining for the past four decades, and overwhelming evidence now links a pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd) to declines across six continents. Because Bd colonizes the skin, antimicrobial peptides secreted onto the surface of the skin are thought to be an important component of the immune defenses against this pathogen. My studies showed that resting frogs constitutively secrete low levels of AMPs that increase following a natural activity such as movement to escape a predator. Using MALDI-TOF MS and growth inhibition assays, I showed that AMPs are present on the skin for at least two hours but are degraded in a time-dependent fashion. Injection of 40 nmol/g norepinephrine results in a long-term depletion of skin peptides. When skin peptides were depleted in juvenile frogs by norepinephrine injection, the frogs were more susceptible to Bd infection and died more rapidly than control frogs with an intact set of skin peptides. These studies support the hypothesis that AMPs are an important defense for this species against chytridiomycosis.

CD4+ T cells developing toward a Th2 fate express IL-4, IL-5, and IL-13 while inhibiting production of cytokines associated with other Th types, such as the Th1 cytokine IFN-γ. IL-4producing Th2 effector cells give rise to a long-lived memory population committed to reactivation of the Th2 cytokine gene expression program. However, reactivation of these effector-derived cells under Th1-skewing conditions leads to cells producing both IFN-γ and IL-4. We now show that this flexibility of cytokine expression is preceded by a loss of the repressive DNA methylation of the Ifng promoter acquired during Th2 polarization. We also demonstrate that flexible expression of Ifng requires the transcription factor STAT4, along with T-bet. Surprisingly, loss of either STAT4 or T-bet increased Ifng promoter CpG methylation in both effector and memory Th2 cells. Taken together, our data suggest a model in which the expression of IFN-γ by Th2-derived memory cells involves attenuation of epigenetic repression in memory Th2 cells, combined with Th1-polarizing signals after their recall activation.

Staphylococcus aureus and Bacillus anthracis are two closely related Gram positive pathogens. The pathogenesis of each of these bacteria often involves a significant blood component. In the blood, both S. aureus and B. anthracis scavenge host heme from hemoglobin in order to satisfy cellular iron requirements. Paradoxically, an overabundance of heme is toxic to these pathogens. Many Gram positive bacteria, including S. aureus and B. anthracis, encode the HssRS two-component signaling system (TCS), which senses heme and regulates hrtAB to protect the bacteria from heme toxicity. Like most TCS, the mechanism of HssRS activation is not well-defined. A chemical genetics approach was undertaken to elucidate the mechanisms by which these pathogens adapt to their host. The Vanderbilt Institute for Chemical Biology small molecule library was screened to identify HssRS-activating compounds. The mechanisms by which lead compounds 882 and 205 activate the heme stress response in S. aureus and B. anthracis, respectively, were dissected. Compound 882 activates S. aureus HssRS by stimulating endogenous heme biosynthesis, indicating that HssRS senses both exogenous and endogenous heme stress. The impact of 882 on heme biosynthesis is mediated by suppressing fermentative processes, functionally connecting heme homeostasis to cellular energy status. Targeting fermentation using the 882 scaffold has therapeutic utility in preventing the outgrowth of antibiotic resistance and reducing S. aureus pathogenesis in vivo. Progress toward identifying the source of 882 toxicity in fermenting S. aureus has pointed to new antibacterial targets. The other compound of interest, small molecule 205, activates hrtAB independent of HssRS, but through a new TCS annotated as BAS1816-17 in B. anthracis. Using 205 as a molecular scalpel, cross-talk between HssRS and BAS1816-17 has been dissected and points to a more complex heme responsive signaling network in the Bacilli. This chemical genetics approach to probing TCS biology has yielded new models for how bacteria regulate heme homeostasis, innovative strategies for targeting bacterial energy production during infection, and a deeper understanding of how bacterial signaling networks are integrated to enable adaptation to complex environments.

This dissertation discusses the role of intersubunit interfaces in HIV-1 capsid assembly and stability. In this study I showed the presence of both the NTD-CTD and CTD-CTD trimer interfaces in mature HIV-1 virions using engineered disulfide crosslinking. Using mutagenesis, I showed that these interfaces are critically important for assembly and stability of the HIV-1 capsid. I also showed that the CTD-CTD trimer interface is formed only after cleavage of CA-SP1. My results provide valuable fundamental knowledge about the biology of capsid assembly and stability and highlight the viral capsid as an attractive target for anti-HIV therapy.

Experimental and epidemiological studies have suggested that the vacuolating cytotoxin (VacA) is an important H. pylori virulence factor that contributes to the development of peptic ulceration and gastric adenocarcinoma. VacA produces a variety of structural and functional changes in eukaryotic cells, many of which are dependent on its ability to form membrane channels. In the absence of a VacA high resolution structure, it has been difficult to analyze VacA structure-function relationships. Thus, one goal of this thesis was to map and characterize VacA functional domains. We describe here the functional characterization of two putative VacA domains (p33 and p55). Collectively, the data generated in this thesis provide strong evidence indicating that both the p33 and p55 functional domains are essential for VacA oligomerization, cell binding, internalization, and vacuolating activity. The second goal of this thesis was to investigate VacA effects on primary human T cells. This study indicates that VacA inhibits activation-induced proliferation of primary human T cells, via a mechanism that requires formation of membrane channels. We propose that the effects of VacA on T cells might contribute to the capacity of H. pylori to evade the adaptive immune response and establish persistent infection.