Salmonella enterica serovar Typhimurium is a common cause of bacterial food poisoning, and S. Typhimurium expresses type 1 fimbriae that enable the bacteria to bind to eukaryotic cells. Fimbrial proteins are encoded by the fim gene cluster (fimAICDHFZYW). The structural components of the fimbriae are FimA (major subunit), FimI, FimH (adhesin), and FimF (adaptor). In order to determine the genes required for fimbrial assembly in S. Typhimurium SL1344, mutations in fimA, fimI, fimH, and fimF were constructed and examined for their ability to produce fimbriae. While SL1344δfimI was able to assemble fimbriae, SL1344δfimA, δfimH, and δfimF were afimbriate, indicating that fimA, fimH, and fimF are each required for fimbrial formation in S. Typhimurium. These results suggest differences in the genetic requirements when comparing S. Typhimurium type 1 fimbrial and E. coli type 1 and Pap fimbrial systems. S. Typhimurium fim gene regulation was also examined. FimZ and FimY are positive regulators of fimbrial gene expression, and FimW is a negative regulator. FimZ is closely related to the family of response regulators of two-component systems. The response regulator activity of FimZ was examined by substituting the conserved aspartate-56 residue, the putative site of phosphorylation, with alanine, to generate an inactive phosphorylation site, or glutamate, to mimic a phosphorylated protein. Resulting strains were examined for fimbrial production and gene expression. It was observed that when the aspartate-56 is substituted with alanine fimbriae are not produced and when glutamate replaces aspartate-56 fimbriae are produced constitutively. Bacterial two-hybrid assays were also carried out to determine the effect of FimZ aspartate-56 substitutions on the previously described FimZ/FimW protein interaction. It was found that FimZD56A is unable to interact with FimW and FimZD56E is able to interact with FimW. Additionally, complementation studies were used to examine the roles of FimZ and FimY in relation to each other and results suggest that FimY acts upstream of FimZ.

Exosomes are cell-derived vesicles that circulate in bio-fluids and enclose cell-associated cargo, producing various consequences in intercellular communication and may contribute to microbial pathogenesis. Exosomes are similar in composition to enveloped viruses, making differentiation between exosomes and enveloped viruses difficult. Yet, exosomes and enveloped viruses may vary considerably in function. Exosomes produced from infected cells may incorporate viral material as they are simultaneously produced with viruses and depending upon the cargo, contribute to disease pathogenesis. Conversely, exosomes from uninfected cells may contribute to protection from infection. Exosomes from breast milk, vaginal fluid, and semen of healthy donors protect against HIV-1. The functional dichotomy of exosomes is unknown. Here, we focus on the function and physical qualities of exosomes found in semen (SE) and how these influence HIV-1. As semen is the major body fluid involved in HIV-1 transmission, exosomes from semen that regulate HIV-1 may contribute to the low incidence of HIV-1 sexual transmission in vivo. Previous studies indicate healthy donor derived SE but not blood exosomes (BE) inhibit HIV-1 in a donor-independent manner. The composition and function of exosomes depends on the status of the exosome-producing cell. Thus, donor characteristics that alter the condition of exosome-producing cells may alter the antiviral phenotype of SE. Illicit drug use enhances HIV-1 replication, negatively affects male fertility, and alters exosome biogenesis pathways. Thus, illicit drugs may alter SE physical, composition, and functional properties. Indeed, SE from donors with a history of illicit drugs were altered in composition which correlated with a diminished ability to inhibit HIV-1. Similarly, because exosomes derived from HIV-1 infected cell cultures promote infection, donor HIV status may contribute to a proviral phenotype of exosomes. Infectivity studies by HIV-infected ART-naïve SE revealed that SE from healthy donors and HIV-infected donors are inhibitory, but BE are not inhibitory. Therefore, the inhibitory phenotype of SE is conserved regardless of donor HIV status. Significantly, BE and SE from HIV-infected ART-suppressed donors not only inhibited HIV-1, but contained inhibitory levels of antiretroviral (ARV) medications, indicating that body-fluid derived exosomes may act as carriers of ARV drugs. Previous studies found that the antiviral mechanism of SE was targeted to multiple HIV-1 lifecycle steps; however, the mechanism of inhibition was unclear. Transcription specific analysis revealed that SE reduced HIV-transcription at multiple steps including association of transcription factors NF-kB and Pol II with the viral promoter, as well as transcription initiation and elongation. SE inhibited HIV-driven promoter activation and viral gene expression. Importantly, SE targeted inhibition of viral protein Tat transcriptional activities. Overall, these findings from donor characteristics that may alter the condition of the cellular source of SE demonstrate that SE antiviral factor(s) is highly conserved. Illicit drugs alter SE-associated factors and may reduce SE inhibitory activities. However, HIV status does not affect the antiviral function of SE. Significantly, donor-ARV medications are associated with SE and BE indicating a potential role of exosomes in drug delivery in vivo. Mechanistically, SE suppress HIV-1 by targeting host and viral transcription factors that could be therapeutically exploited for novel anti-HIV strategies. These data emphasize the need for additional studies on the composition and function of SE to harness the antiviral potential of SE inhibitory factors.

No description available.

Tumor necrosis factor (TNF) is a pleotropic cytokine that mediates many inflammatory and innate immune responses. TNF also causes apoptosis in certain transformed cell lines and cells that are infected with certain viruses or intracellular bacteria. Cytosolic phospholipase A2 (cPLA2) is an inflammatory enzyme that mediates its activities, by specifically catalyzing the release of arachidonic acid leading to the generation of eicosanoids. The activity of cPLA2 is necessary during TNF-induced apoptosis and the goal of this study was to identify signals that mediate the activation of cPLA2 during cell death. Intracellular calcium and phosphorylation are well documented to activate cPLA2 under many inflammatory conditions. Therefore, I examined the ability of these signals to regulate cPLA2 during TNF-induced apoptosis. I first examined calcium levels during the TNF-induced apoptosis of C3HA fibroblasts and determined that an influx of extracellular calcium occurs early during cell death. This influx, as well as the release [3H]arachidonic acid, was blocked by verapamil indicating that the calcium response is necessary for the activation of cPLA2 during this process. To analyze the effects that phosphorylation has during TNF-induced apoptosis, cPLA2 proteins, containing serine phosphorylation site mutations, were stably overexpressed in WM793 melanoma cells. Although PMA was able to enhance the release of [3H]arachidonic acid from cells that overexpressed cPLA2, the treatment of the same cells with TNF and cycloheximide had no effect. However, subsequent experiments using PMA demonstrated novel roles for the phosphorylation of Ser-437 and ?727. One was an activation role for Ser-437 as its phosphorylation enhanced [3H]arachidonic acid release. The other was an inhibitory role for the phosphorylation of Ser-727 as its mutation suppressed the release in response to PMA. In conclusion, though the activation of cPLA2, by calcium responses, during apoptotic and non-apoptotic systems is consistent, the regulation of cPLA2 by phosphorylation may involve both positive and negative regulatory signals.

The anaerobic hyperthermophilic archaeon, Pyrococcus furious, possesses a system for the detoxification of reactive oxygen species, which is different from the classical defense mechanisms present in aerobes. P. furiosus employs a novel enzyme system centered on the enzyme superoxide reductase (SOR), which reduces superoxide molecules to hydrogen peroxide without producing oxygen. Surprisingly, P. furiosus SOR, unlike many P. furiosus enzymes, was shown to function at low temperature (<25o C). A model for superoxide reduction by SOR was proposed where the electrons used by SOR to reduce superoxide are supplied by a small iron containing protein, rubredoxin (Rd), and Rd is reduced by the oxidoreductase, NAD(P)H-rubredoxin oxidoreductase (NROR). The first objective of this study was to evaluate the validity of the proposed superoxide reduction pathway by using the recombinant SOR, Rd and NROR enzymes in an in vitro assay as well as to demonstrate in vivo function via complementation studies in superoxide detoxification deficient Escherichia coli strains. The second objective was to investigate the transcriptional expression levels of genes that are involved in the SOR-centered superoxide reduction pathway in order to determine how these genes are expressed and regulated in response to various oxidative stresses. The third objective was to evaluate the efficacy of the biotechnological application of this superoxide detoxification system by expressing SOR in plant cells, which enhanced their survival at high temperature and from drought indicating that it functions successfully in vivo. The fourth objective of this study was the characterization of glutathione reductase (GR) from a psychrophile, Colwellia psychrerythraea, which is stable at low temperatures and protects cells from free radicals by serving as a reductant. The C. psychrerythraea GR gene was cloned into an E. coli-based recombinant expression system. Recombinant C. psychrerythrae GR was expressed and purified. The recombinant GR showed significant activity at low temperature (4C). The P. furiosus superoxide reduction system genes and GR from C. psychrerythraea can be engineered into plants (Arabidopsis) to aid in combating damage caused by oxidative stress when plants undergo rapid changes in temperature, high light or UV exposure, or drought conditions.

The majority of Listeria monocytogenes isolates from foods and the environment are serotype 1/2a strains. However, serotype 4b strains cause the majority of human listeriosis outbreaks. The purpose of the research has been to compare the growth of serotype 1/2a strains and serotype 4b strains in biofilms. A method to enumerate viable L. monocytogenes cells of each serotype in mixed culture biofilms was developed using real-time PCR with propidium monoazide. To determine the competitive fitness of strains of serotype 1/2a and 4b, cocktails of each serotype were mixed to form biofilms. The biofilms were treated with a simulated food processing (SFP) system composed of repeated cycles of growth, sanitation treatment, and starvation. Data show that the serotype 1/2a strains were generally more efficient than the 4b strains at forming biofilms and predominated in the mixed culture biofilms. The growth of 4b strains was not inhibited in mixed culture biofilms compared to the single serotype (4b) biofilms in the SFP system. To compare the density of biofilms formed by strains of the two serotypes, 18 strains of each serotype were examined for biofilm formation under a variety of conditions, including varying concentrations of glucose, sodium chloride and ethanol at different temperatures using a microplate assay. Results indicate that the serotype 1/2a strains formed higher density biofilms than the 4b strains under most conditions. The data from this project support the hypothesis that L. monocytogenes serotype 1/2a strains are more efficient in biofilm production than 4b strains and may help to explain the higher percentage of 1/2a isolates from foods and the environment.

Viral myocarditis may be caused by a number of different viruses, and the mechanisms of pathogenicity can be both immune mediated and/or due to direct cytopathic effect. While extensive research has been done regarding immune-mediated mechanisms, the mechanisms of direct cytopathic effect remain largely unexplored. Reovirus induced murine myocarditis is not immune mediated, providing for an excellent disease model. Reovirus induction of, and sensitivity to, interferon-beta (IFN-ß) has previously been found to be an important determinant for protection against viral myocarditis. There are many interferon regulatory factors that can both regulate, and be regulated by, IFN-ß. In the first part of this dissertation, transient transfections of primary cardiac myocyte cultures (PCMCs) were used to investigate the role of interferon regulatory factor-1 (IRF-1) in regulation of IFN-ß. We found that while IRF-1 is downstream of the IFN response, it still plays an important protective role against viral myocarditis. In the next chapter, reovirus induction of Interferon Stimulated Genes (ISGs) was investigated in PCMCs and compared with a more generalized cell type, primary murine embryonic fibroblasts (PMEFs). Here we found that reovirus induction of ISGs is cell type-specific and interferon-mediated, yet levels of induction are discordant with viral induction of IFN. These results suggested that cell types that are prone to induction of IFN are resistant to induction of ISGs. In appendix I, reovirus induction of an Interferon Stimulated Response Element (ISRE) was investigated in the above mentioned cell types, providing insight into reovirus induction of ISGs and the cell type-specific response. In appendix II, the effect of IRF-1 on ISG induction was further examined by overexpressing IRF-1. These data indicated that IRF-1 could regulate ISGs and ISRE sequences, but that IFN may induce or activate a product that inhibits IRF-1 induction of these genes. In the final appendix, reovirus induction of the ISG, IRF-7, was investigated in PCMCs and PMEFs using real-time PCR. Here, we found that reovirus induction of IRF-7 is discordant with reovirus induction of IFN. Possible mechanisms of differential ISG induction are discussed in the summary of this dissertation to provide an overall picture of the cardiac IFN response to virus infection.

Viral myocarditis is an important human disease associated with a wide variety of viruses. The cardiac damage and inflammation associated with viral myocarditis can be immune mediated and/or due to direct cytopathic effect. Reovirus-induced myocarditis reflects direct virus-mediated apoptosis of cardiac cells, providing an excellent model to study direct cytopathic effect in the heart. Previous work has found interferon-beta (IFN) to be an important determinant for protection against viral myocarditis. Importantly, IFN signals through the Jak-STAT pathway to induce the expression of interferon-stimulated genes (ISGs) and establish an antiviral state. First, we investigated the underlying mechanisms of the IFN response in these cardiac cells. We found that high basal IFN-beta expression in cardiac myocytes protects this vulnerable, nonreplenishable cardiac cell type, while high basal expression of interferon alpha receptor 1 and latent Jak-STAT components in adjoining cardiac fibroblasts renders these cells more responsive to IFN and prevents them from inadvertently serving as a virus reservoir. This provides the first evidence for an integrated network of cell-type-specific innate immune components for organ protection. Next, we investigated whether particular reovirus strains were able to repress IFN signaling. We found that the mildly myocarditic reovirus, T1L, but not the nonmyocarditic reovirus, T3D, could repress IFN induction of ISGs and that this repressor function is determined by the M1 gene of T1L. Infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of IRF9 in the nucleus; an effect not previously described for any virus. The M1 gene has also previously been identified as a determinant of virus strain-specific differences in the IFN response, and the M1 gene and the IFN response have been identified as determinants of virus strain-specific differences in induction of murine myocarditis. Here, we find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response, and provide evidence that virus strain-specific differences in IFN antagonism are a determinant of disease.

The AL2 protein of tomato golden mosaic virus (TGMV) is multifunctional. It is required for derepression of the TGMV AR1 gene in phloem tissue, and for trans-activating the AR1 and BR1 genes in mesophyll cells. It also enhances virus susceptibility when expressed in transgenic plants. It is thought that TGMV AL2 protein accomplishes these functions by interactions with unknown host factors. In this study, cDNAs from Nicotiana benthamiana plants that encode proteins which interact with TGMV AL2 were characterized. A yeast two-hybrid assay identified two cDNA clones, Nb#51 and Nb#62, that specifically interacted with TGMV AL2, but not with negative control proteins. Sequences of these two cDNA clones were determined by primer walking, which revealed that Nb#51 appears to be a 3?-coterminal truncated version of Nb#62. Inspection of the amino acid sequences encoded by Nb#62 found the presence of both ankyrin-repeats and tetratricopeptide repeats (TPR). Deletion analysis showed that the TPR motif, together with its flanking regions, was sufficient to confer on Nb#62 the ability to interact with TGMV AL2, whereas the ankyrin-repeats were not required for this interaction. Nb#62-specific mRNAs were detected in N. benthamiana plants by northern hybridization in potato virus X (PVX) infection experiments, but not in heat-shock experiments. Virus-induced gene silencing (VIGS) assays were used to investigate the possible function(s) of the Nb#62-encoded protein in normal plants and in the context of a TGMV infection. When PVX carrying a 618-bp fragment from the 5?-end of the Nb#62 cDNA was used as a silencing trigger in N. benthamiana plants, VIGS effectively targeted the transcripts which contained sequence similarity with the trigger fragment. However, the infected plants didn?t have difference in the phenotype when compared to the PVX vector infection. When TGMV carrying a 93-bp fragment from the 5?-end of the Nb#62 cDNA infected plants, viral DNA accumulation in the upper leaves was reduced, when compared to wild-type TGMV or a control TGMV construct containing a 95-bp fragment from the tobacco Sulfur gene.

V(D)J recombination in developing lymphocytes is essential for producing a diverse repertoire of antigen receptors (TCR and Ig). During recombination, the proteins encoded by the recombinase activating genes (RAG1 and RAG2) bind specific DNA sequences flanking individual V, D, and J coding segments within each antigen receptor gene, and introduce double strand DNA breaks at the coding sequence/targeting sequence boundaries. These double strand breaks are then repaired by ubiquitous DNA repair machinery to generate novel coding segment joints. The ability of each developing lymphocyte to independently assemble unique V(D)J joints results in the enormous diversity of antigen receptors expressed by our immune system. Despite a conserved enzymatic activity in both B and T lymphocytes, the assembly of T cell receptors (TCRs) and Immunoglobulins (Igs) in T and B cells respectively follows a highly orchestrated program in which the accessibility of individual targeting sequences varies during lymphocyte development. For example, when the TCRb locus is rearranged, it initially assembles joints between D and J elements. Only after DJ joining do upstream V sequences become accessible and rearrange with the preassembled DJ?s. We have previously shown that DJ rearrangement requires modification of the chromatin structure surrounding individual D and J segments via the coordinated actions of D-associated promoters and a single downstream enhancer. Like the D elements, each V element in TCRb is associated with a transcriptional promoter. But the role these V promoters play in V-to-DJ recombination remains unknown. Similarly, because enhancer deletion ablates D-to-J assembly, the potential role of enhancer activity in V recombination has not been directly tested. We hypothesize that V-to-DJ rearrangement requires both enhancer and promoter-dependent changes in the chromatin surrounding the V RAG binding site, as well as that surrounding the D 5? binding site. To test this hypothesis, I have constructed a panel of recombination substrates which harbor unrearranged or prerearranged DJ elements downstream from a single V element. These ?miniloci? were stably transfected into the chromatin of a recombinase-inducible T cell line, and the chromatin status, expression and recombination potential of each was assessed.