Mycobacterium tuberculosis is responsible for nearly three million human deaths every year. Understanding the mechanisms and bacterial factors responsible for M. tuberculosis' ability to cause disease in humans is critical for the development of improved treatment strategies. Using negative staining and transmission electron microscopy it was discovered that mycobacteria, including the human pathogen M. tuberculosis, produce fine surface structures known as pili. Mass spectroscopy analysis demonstrated that purified pili from M. tuberculosis are comprised of protein subunits encoded by the predicted M. tuberculosis H37Rv ORF designated Rv3312A. These pili termed M. tuberculosis pili, Mtp, are highly aggregative 2-5 nm diameter fibers and are recognized by IgG antibodies contained within TB patient sera. These results indicate that Mtp are produced during human infection. Mtp bind to the extracellualr matrix protein laminin in vitro suggesting that Mtp are a newly identified adherence factor for M. tuberculosis.A second pili morphotype that appeared as rope-like bundles were observed for M. tuberculosis and it was found that the M. tuberculosis chromosome contains a type IVB pili gene cluster. The M. tuberculosis type IV pili belong to the Flp sub-family of type IVB pili. RT-PCR analysis reveals that flp is expressed by M. tuberculosis and IF microscopy with Flp-specific antibodies shows the Flp protein is secreted from the bacteria. Evidence presented herein also demonstrates that an Flp-derived peptide is capable of polymerizing into pili-like fibers in vitro over a pH range of 4.5-7.5. Further studies show that the M. tuberculosis type IV pili are encoded by a novel 5-kb genomic island that contains the flp prepilin and putative biogenesis genes. The flp genomic island is characterized by an increased G+C content of 70% (the mean G+C content of the M. tuberculosis chromosome is 65%) and is flanked by multiple direct repeats. The identification of type IV pili in M. tuberculosis is the first report of any classical virulence factor for the bacillus and the genetic characteristics of the locus strongly suggest this chromosomal region was horizontally acquired.

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No description available.

Staphylococcus aureus is a major pathogen, which causes a wide range of infections. Despite its obvious clinical importance, little is known about the mechanisms of pathogenesis. An in vitro model mimicking infection was developed in order to identify putative virulence determinants. The model involves the growth of S. aureus in serum under microaerobic conditions. All known virulence factors tested were shown not to be required for growth, or preferentially expressed, in serum. Tn917 transposon libraries of S. aureus were screened to identify genes preferentially expressed in serum, compared to a nutrient-rich growth medium. 73 clones were identified and the transposon insertion site was characterised for 23 of these clones. Analysis of sequence flanking the transposon insertion revealed the identity of the mutated loci. 10 out of 23 sequenced clones, contained transposons inserted within genes involved in the biosynthesis of the aspartate family of amino acids (lysine. threonine, methionine and isoleucine). These were: the two common pathway enzymes; aspartokinase (lysC) , and aspartate semi aldehyde dehydrogenase (asd) , along with; dihydrodipicolinate dehydrogenase (dapA), and cystathionine y-synthase (yjcf) , involved in the biosynthesis oflysine and methionine respectively. Analysis of methionine biosynthesis indicated that S. aureus possesses only a single pathway, which proceeds via cystathionine. Several genes encoding methionine biosynthetic enzymes were found clustered on the S. aureus chromosome. The genes lyse, asd and dapA were found to be encoded by the first three genes of an eight gene operon, which also contains three other genes involved in lysine biosynthesis. This operon named the dap operon, is the major lysine biosynthetic operon of S. aureus. lysC, asd and dapA were all found to be repressed at the transcriptional level primarily by lysine, although factors other than the availability of lysine may be responsible for the regulation of lysine biosynthetic gene expression in serum. lysC, asd and dapA were all found to be expressed in vivo, in a murine pyelonephritis model using both RT-PCR and TaqMan techniques. However, these genes were not found to be important in three murine pathogenicity models. Finally, in addition to the development of a model mimicking infection, and the identification of genes with a potentially important role in vivo, this thesis has enhanced our understanding of both methionine and lysine biosynthesis in S. aureus.

Abstract not available

Temperature is a critical environmental signal, which exerts powerful control over the development and virulence of diverse microbial pathogens. Fungi, along with other microbial species, exploit a diversity of mechanisms to sense and respond to temperature fluctuations that may be encountered in the host or under other conditions of temperature stress. For Candida albicans, the leading fungal pathogen of humans, temperature influences mating, phenotypic switching, resistance to antifungal drugs, and the morphogenetic transition from yeast to filamentous growth. C. albicans morphogenesis is strongly influenced by temperature, and most filament inducing cues depend on a concurrent increase of temperature to 37˚C before morphogenesis can occur. Further elevated temperature of 39˚C to 42˚C can serve as an independent filament-inducing cue, although the molecular mechanisms underpinning this temperature-dependent morphogenetic transition remain largely uncharacterized. Here, I provide the first comprehensive investigation of the molecular mechanisms mediating temperature-dependent morphogenesis in C. albicans. I establish that the thermally responsive molecular chaperone Hsp90 orchestrates temperature-dependent morphogenesis, and that Hsp90 functions as a key temperature sensor, such that elevated temperature is required to relieve Hsp90-mediated repression of the morphogenetic program. Further, I demonstrate that Hsp90 controls morphogenesis via at least two distinct cellular signaling cascades. First, Hsp90 and its co-chaperone Sgt1 physically interact, and together regulate protein kinase A (PKA) signaling via an interaction with the adenylyl cyclase of the PKA cascade, Cyr1, such that genetic depletion of either Hsp90 or Sgt1 activates PKA signaling and induces filamentation. Second, Hsp90 controls temperature-dependent morphogenesis via previously uncharacterized cellular circuitry comprised of the cyclin-dependent kinase Pho85, the cyclin Pcl1, and the transcriptional regulator Hms1. Together, this research illuminates the central role of Hsp90 in coupling temperature sensing and morphogenesis in the human fungal pathogen C. albicans.

The influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity. Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown. The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events. Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis. The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor. After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways. This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

NK cell lymphoma is one of the cellular malignancies that arise from lymphocytes. Due to its rarity and aggressiveness the detailed molecular pathogenesis of NK cell lymphoma remains to be discovered. There are recent studies showing that the master regulator of B-cell differentiation into plasma cells, the Positive Regulatory Domain containing 1, with ZNF domain(PRDM1) has tumor suppressive function not only in diffuse large B-cell lymphoma (DLBCL), but also in NK cell lymphoma. The PRDM1 has two isoforms, αand β, where the former one is a functional isoform and the latter is a defective isoform with shortened and disrupted positive regulatory domain formed from transcription of internal promoter. By semi -quantitative RT-PCR, PRDM1-αexpression was found to be absent in 80% (4/5) NK cell lines while present in the normal NK cells. Loss of PRDM1 expression suggests its role as tumor suppressor. In order to study the tumor suppressive role of the αisoform of PRDM1, short-hairpin RNA (shRNA) with isoform specific sequence is used to knockdown the expression of PRDM1-αin NK cell lines. Western blot result showed about 40% decrease of PRDM1-αprotein after knockdown. Retroviral infection of the NK cell lines, NKYS and YT which have endogenous α-isoforms of expression, for the delivery of the shRNA was done and were subsequently subjected to in vitro functional analyses including MTS assay, colony formation assay, cell viability test and cell cycle analysis to determine potential effect of the loss of PRDM1-αon the NK cell lines. The PRDM1-αprotein isoform is expected to be able to repress excessive growth of NK cell line. When this isoform is inactivated, the NK cell lines are expected to proliferate significantly than the negative control counterpart in functional analyses. However in this study only YTcell line showed significant proliferation advantage in MTS and colony formation assay after the knockdown of PRDM1-α by shRNA. Cell viability assays and cell cycle analyses failed to show significant changes in both NK cell lines and yet even showed inhibitory effect after the knockdown of the gene. Ectopic expression of PRDM1-αby retroviral infection was done in KHYG cell line to further evaluate its tumor suppressive function. Apoptotic assay on the KHYG cells with ectopic expression of PRDM1-αwas performed and percentage of cells with late apoptosis was found to be significantly higher in this cell line. This suggests that one of the mechanisms for PRDM1-αto act as tumor suppressor is via the apoptosis pathway which in turn promotes the cell death. Future studies will be made to further investigate the effects of knockdown of PRDM-1αby designing another shRNA sequence which knockdown the expression of gene by at least 50% and to further investigate the role of PRDM1-αinthe pathogenesis NK cell lymphomaby proliferation assays, colony formation assay and cell cycle analysis. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, characterized by the production of autoantibodies and immune-mediated injury to the kidney. Hyaluronan (HA) is a major component of the extracellular matrix, which is involved in immune-mediated renal injury. We have previously demonstrated that HA expression is increased in the glomerulus of patients with severe lupus nephritis, attributed in part to anti-dsDNA antibody-mediated induction of low molecular weight (LMW) HA and high molecular weight (HMW) HA synthesis in mesangial cells. The causal role of HA and its fragments in the pathogenesis of lupus nephritis has not been explored. In this project, two separate in vivo studies were undertaken to delineate the role of HA in disease progression in NZBWF1/J mice. In the first study, we determined the effect of 4-methylumbelliferone (MU), a specific inhibitor of HA synthesis, on renal function and histology in NZBWF1/J mice with active disease, with particular emphasis on inflammatory and fibrotic processes. In the second study, we investigated whether exogenous HA could exacerbate disease manifestations in pre-disease NZBWF1/J mice. The mechanisms underlying the effects of MU and exogenous LMW HA and HMW HA were investigated in mesangial cells isolated from NZBWF1/J mice. Female NZBWF1/J mice with established nephritis were randomized into 3 groups and treated with (1) PBS, (2) Arabic gum (Gum) or (3) MU (3g/kg/day) for 2, 4, 8 and 12 weeks. Treatment of mice with MU for 12 weeks reduced serum HA levels and abrogated intra-renal expression of HA compared to PBS and Gum treated mice. Inhibition of HA synthesis in the kidney resulted in decreased IgG and C3 deposition, reduced B cell, T cell and macrophage infiltration, matrix accumulation and TNF-, IL-6 and MCP-1 expression, which was associated with improved renal functions. To confirm that HA influenced pathogenesis of lupus nephritis, pre-disease NZBWF1/J mice were randomized to receive (1) PBS, (2) LMW HA or (3) HMW HA for periods up to 24 weeks. Administration of LMW HA and HMW HA into NZBWF1/J mice by tail-vein injection induced intra-glomerular deposition of IgG and C3, B cell infiltration, glomerular hypercellularity and tubular atrophy, which was accompanied by induction of MAPK signaling pathways, enhanced MCP-1 expression, and increased matrix deposition in the glomerular and tubular basement membranes. In vitro studies showed that exogenous IL-6, IL-1, TGF-1 and TNF- induced HA synthesis in murine mesangial cells (MMC), with over 80% secreted into the conditioned medium. This was accompanied by an increase in pro-inflammatory cytokine secretion and synthesis of fibronectin and laminin. Inhibition of HA synthesis with MU significantly decreased cytokine secretion and fibronectin synthesis. The ability of HA to induce inflammatory and fibrotic processes in mesangial cells was confirmed in separate studies in which MMC were incubated with exogenous LMW HA and HMW HA. In summary, these original findings provide evidence of a direct effect of HA on intra-renal inflammation and fibrosis in lupus nephritis. Approaches to inhibit HA synthesis may offer novel therapeutic strategies to delay disease progression. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Background and Objectives: Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells. The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D. Key findings: 1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples; 2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients; 3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis; 4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D. Conclusions: Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy