Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Herein, we describe a mass spectrometry-based approach to identify a T4SS subassembly that contains CagL, CagH, and CagI. We demonstrate that these three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Moreover, we show that these proteins play key roles in biogenesis of T4SS pili at the bacteria-host cell interface. Collectively, these results highlight the important role played by unique constituents of the H. pylori cag T4SS, and illustrate the marked variation that exists among bacterial T4SSs.

Populations of amphibians around the world have experienced devastating declines in the last few decades. A newly emerging fungal pathogen, Batrachochytrium dendrobatidis, has been implicated as one cause of these declines. Some frog species, such as Xenopus laevis, are considered resistant to lethal infections of B. dendrobatidis, whereas other species are susceptible. My work focused on whether innate or adaptive immune defenses, or both, play a role in determining a species overall resistance to fatal B. dendrobatidis infections. My research concluded that innate antimicrobial peptide defenses in the skin play a critical role in resistance, as does an irradiation-sensitive component of the adaptive immune system. However, further research found that B. dendrobatidis has the ability to attenuate adaptive responses by preventing proliferation of activated lymphocytes via the induction of apoptosis in these cells. My overall work suggests that the interplay between the strength of both innate and adaptive immune responses after B. dendrobatidis exposure and the capacity of B. dendrobatidis to impair adaptive immunity dictate a species overall resistance to B. dendrobatidis infections.

HIV-epitope-specific T cell responses are critical components of the natural immune response to HIV infection, but these cells often become dysfunctional in chronic infection. Structural diversity within the epitope-specific T cell receptor (TCR) repertoire is likely beneficial in the suppression of viral epitope variants. However, the relationship between the structural and clonotypic composition of the HIV-specific TCR repertoire, clonotypic surface and functional phenotype, and avidity for and exposure to antigen is poorly defined. Dominant and sub-dominant epitope-specific T cell clonotypes were identified and the TCR repertoire was assessed over time. Surface expression of PD-1, CD38, CD127, CD45RO, and CCR7 was measured and used to define exhaustion and memory profiles on epitope-specific T cell populations during chronic infection and after initiation of antiretroviral therapy. Dominant clonotypes in chronic infection express a surface phenotype consistent with exhaustion which is unchanged after narrowing of the repertoire following initiation of antiretroviral therapy. Despite their cell surface phenotype, dominant clonotypes were capable of cytokine production and proliferation when stimulated with peptide epitopes, but sub-dominant clonotypes produced higher relative levels of cytokines and displayed greater variant cross-recognition compared to corresponding dominant clonotypes. Together, these data indicate that dominant clonotypes within HIV-specific T cell responses display a phenotype consistent with ongoing exposure to cognate viral epitopes, that TCR repertoire diversity may be maintained by antigen exposure, and suggest that cross-reactive, sub-dominant clonotypes may retain greater capacity to suppress replication of viral variants. Overall, these findings impact our understanding of how the TCR repertoire is maintained in HIV and provide insight into the potential effects of future immunomodulatory therapies.

MICROBIOLOGY AND IMMUNOLOGY HUMAN HEMOGLOBIN AS AN IRON SOURCE OF STAPHYLOCOCCUS AUREUS GLIB PISHCHANY Dissertation under the direction of Professor Eric P. Skaar Staphylococcus aureus is a tremendous human pathogen that causes an array of life-threatening diseases. During invasive infection, S. aureus employs the iron regulated surface determinant (isd) system to bind and utilize host hemoglobin as a primary source of nutrient iron. We have found that surface anchored proteins of the Isd system co-localize within the cell wall. These results contribute to a mechanistic understanding of nutrient transport across the Gram positive cell wall. Further, we demonstrate that S. aureus has evolved to recognize human hemoglobin with improved efficiency compared to hemoglobin derived from other animal species. This evolutionary adaptation substantially contributes to pathogenicity, as revealed by an increased susceptibility to staphylococcal infection of transgenic mice expressing human hemoglobin. These results establish an improved animal model of systemic staphylococcal infection, and suggest that polymorphisms within human hemoglobin may affect individual susceptibility to S. aureus.

Cytoplasmic antigens derived from viruses, cytosolic bacteria, tumours and allografts are presented to T cells by MHC class I or class II molecules. In the case of class II-restricted antigens, professional antigen-presenting cells acquire them during uptake of dead, class II-negative cells and present them via a process called indirect presentation. It is generally assumed that the cytosolic, or class I-processing machinerywhich supplies peptides for presentation by class I moleculesplays very little role in indirect presentation of class II-restricted, cytoplasmic antigens. Remarkably, upon testing this assumption, proteasomes, TAP and ERAAP, but not tapasin, partially destroyed or removed cytoplasmic, class II-restricted antigens such that their inhibition or deficiency led to dramatically increased Th cell responses to cytosolic allograft (HY) and microbial (Listeria monocytogenes) antigens, and uncovered novel class II-restricted self-epitopes and those derived from SV40 large T antigen. From these findings, and the altered repertoire of class II-restricted self-peptides presented by wild type, TAP- and ERAAP-deficient cells, a novel model emerges in which the class I-processing machinery regulates the quantity, as well as quality of cytoplasmic peptides available for presentation by class II molecules, and hence modulates TH cell responses, as well as Th T cell receptor repertoire.

APOBEC3G (A3G) is a cytidine deaminase that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in the absence of the HIV-1 virion infectivity factor (Vif) protein. However, in the presence of the viral Vif protein, A3G is targeted for proteasomal degradation. If not degraded, A3G is packaged into progeny virions where it inhibits early steps of HIV-1 replication in the target cell. This work identifies and characterizes the ability of A3G to restrict late steps of HIV-1 replication in the producer cell. A3G is found in two forms in cells: in a diffuse Low Molecular Mass (LMM) form and a large High Molecular Mass (HMM) form. We call the HMM form A3G Complexes. We show that A3G complexes partially co-localize with RNA granules and decrease HIV-1 production. This restriction of virus production exists only when A3G is found within complexes. A3G complexes shorten the half-life of an intracellularly-retained Gag protein. Different late HIV-1 replication steps are restricted by RNA granules that contain A3G versus those without A3G. This work will lead to better understanding of the biology of A3G complexes and RNA granules as they relate to HIV-1 replication.

The ArsRS two-component system (TCS), comprised of a sensor histidine kinase (HK) ArsS and a response regulator (RR) ArsR, contributes to pH-responsive gene regulation in Helicobacter pylori. In a typical TCS, a specific environmental stimulus triggers the HK to phosphorylate and induce a conformational change in the RR, altering its cellular functions. In order to elucidate structure-function relationships within the ArsRS TCS, we expressed and purified the full-length ArsR protein and its DNA-binding domain (ArsR-DBD), and analyzed the tertiary structure of ArsR-DBD using solution nuclear magnetic resonance (NMR) methods. The structure of ArsR-DBD consists of an N-terminal four-stranded beta-sheet, a helical core, and a C-terminal beta-hairpin. The overall tertiary fold of ArsR-DBD is most closely related to DBD structures of the OmpR/PhoB subfamily of bacterial RRs. To identify ArsR protein elements that potentially bind target DNA sequences and thereby regulate gene transcription in H. pylori, we modeled ArsR-DBD in complex with a putative DNA target. Additionally, we identified novel members of the ArsRS regulon by analyzing ArsR binding to the promoter sequences of putative target genes. These results broaden our understanding of the ArsRS TCS and its role in regulating gene expression in H. pylori.

In metazoans, developmentally regulated genes depend upon cis-regulatory elements for cell-type specific and heritable transcription. By employing a BAC transgenic system, I demonstrate the function of the noncoding region of the human interferon-gamma (IFNG) locus. Deletion of individual IFNG regulatory elements results in cell-type specific loss of gene expression, stimulus-specific gene up regulation, or loss of expression in suboptimal conditions. These results demonstrate that appropriate cell and stimulus specific expression of IFNG is achieved through the interactions of multiple unique distal regulatory elements.

Virulence effectors are a diverse group of proteins secreted by pathogenic bacteria to enable infection by concertedly acting to obstruct host defenses. A major step during invasion and spread for intracellular pathogens and during attachment for adherent pathogens is to alter the host cell cytoskeleton. A combination of structural and functional studies was used to probe the roles of two effectors in cytoskeleton rearrangement during pathogenesis of E. coli and Shigella. Structural studies indicate that EspG from E. coli and VirA from Shigella are structurally similar proteins, yet functional studies indicate that they have different cellular targets. EspG can functionally substitute for VirA, suggesting that these effectors target the same cellular pathway and likely bind similar ligands. Our data indicate that both effectors promote actin reorganization rather than catalyze microtubule destruction as originally proposed.

The process by which viruses cause disease consists of a complex series of steps involving many host and viral factors. The first step in the viral replication cycle, attachment of the virus to cell-surface receptors, is important for establishing infection and serves a critical function in viral pathogenesis. Mammalian reoviruses, a highly tractable experimental model for the study of viral pathogenesis, display serotype-specific patterns of tropism and disease within the murine central nervous system (CNS) attributable to the viral attachment protein, σ1. While all reovirus serotypes engage the proteinaceous receptor junction adhesion molecule-A, these strains differ in carbohydrate coreceptor utilization, suggesting that carbohydrate binding by σ1 influences reovirus pathology in the CNS. However, mechanisms underlying reovirus spread to and within the CNS are not well understood. The goals of my research were to elucidate the molecular basis of sialic acid (SA) binding by T3 reovirus and define the contribution of SA engagement in reovirus target-cell selection and neuropathogenesis. First, I report the crystal structure of T3 σ1 in complex with sialylated oligosaccharides. Using structural information and mutagenesis studies, I defined a discrete network of residues in T3 σ1, centered on Arg202, that are required for SA binding. Furthermore, data gathered from in vivo experiments comparing strains that differ in the capacity to bind SA suggest that SA-binding enhances reovirus neurovirulence by allowing more efficient infection of CNS tissues. These findings reveal the structural and molecular basis of T3 σ1-SA interactions and suggest that SA-binding enhances reovirus infection of the murine CNS. Results from this research have enhanced an understanding of mechanisms of reovirus attachment to neuronal cells and the molecular interactions and structural features that mediate receptor engagement by viral attachment proteins. Knowledge of pathologic mechanisms gained from this research may establish general principles of viral neuropathogenesis, thereby improving our ability to develop antiviral therapeutics. Moreover, an enhanced understanding of the molecular interactions that occur between σ1 and its cellular receptors will aid in the continued development of reovirus as an oncolytic agent and vaccine vector.