Browsing by Subject "Cell wall"
- Results Per Page
- Sort Options
Item Open Access Cellular Coordinators: Mechanisms by Which Non-Enzymatic Proteins Contribute to Growth and Cell Surface Remodeling in the Human Fungal Pathogen Cryptococcus neoformans(2022) Telzrow, Calla LeeMy thesis work has focused on characterizing mechanisms by which human fungal pathogens regulate their adaptive cellular responses in order to survive and cause disease in the human host. Unlike most microbial fungi found in the environment, Cryptococcus neoformans has become a successful human pathogen due to two intrinsic abilities: 1) to survive and grow at human body temperature and 2) to employ virulence factors to combat host immune defenses. Over the past two decades, the fungal pathogenesis field has made enormous progress in identifying and characterizing C. neoformans proteins responsible for these adaptive cellular responses with a particular focus on enzymes, like those involved in cell cycle progression or those responsible for synthesizing components of the fungal cell surface. Although we know a substantial amount about the functions of these enzymes and their implications on fungal pathogenesis, the mechanisms by which these enzymes are regulated are less clear. I have attempted to address this gap in knowledge by focusing my thesis work on the identification and characterization of C. neoformans non-enzymatic proteins that regulate enzymes important for adaptive cellular responses. I have identified and characterized the C. neoformans arrestin proteins as regulators of enzyme ubiquitination, and likely enzyme function, in response to specific extracellular stressors (Chapters 2 & 3). I have also characterized a Cryptococcus-specific protein, Mar1, as an important modulator of host-fungal interactions due to its regulation of cell surface remodeling through maintenance of mitochondrial metabolic activity and homeostasis in response to cellular stress (Chapters 4 & 5). Furthermore, I also performed a comprehensive comparative analysis of different RNA enrichment methods for RNA sequencing applications and long non-coding RNA identification in C. neoformans, which can help researchers select appropriate tools for studying adaptive cellular responses from the RNA level (Chapter 6). These studies collectively have demonstrated that non-enzymatic proteins are important “cellular coordinators” in human fungal pathogens; they regulate the activity of many different enzymes in response to distinct extracellular signals, and as a result are required for both fungal growth and virulence factor employment in response to host-relevant stressors.
Item Open Access Mechanisms of Protein Localization in Cryptococcus neoformans Mediate Virulence and Immune Recognition(2018) Esher, ShannonCryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease and death in immunocompromised populations, in particular among those with advanced HIV infection. This fungus is found ubiquitously in the environment and acquired through inhalation into the respiratory tract followed by dissemination to the central nervous system in immunocompromised individuals. The ability for C. neoformans to sense and adapt to the host environment is crucial to its success as a pathogen. Many C. neoformans proteins require proper subcellular localization for their function, and as such this fungus carefully regulates the localization of proteins involved in important cellular processes related to host adaptation.
Fungal growth and morphogenesis, as well as thermotolerance and virulence are controlled by conserved Ras-like GTPases. These proteins require proper localization for full function and are directed to cellular membranes through the posttranslational modification process known as prenylation. Using the tools of fungal genetics and molecular biology, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase -subunit is required for thermotolerance and pathogenesis. We also identified and characterized post-prenylation protease and carboxyl methyltransferase enzymes in C. neoformans, demonstrating that these later steps have only subtle effects on stress response and fungal virulence. By fluorescent microscopy and molecular biology, we show that Ram1 is required for proper subcellular localization of Ras1, but not Cdc42, and that the post-prenylation processing steps are dispensable for the localization of these substrate proteins.
C. neoformans dramatically alters its cell wall upon entering the host in order to facilitate immune avoidance. Using the tools of forward genetics, we identified a novel cell wall regulatory protein, Mar1. We have demonstrated that this protein is required for capsule attachment and full virulence in mouse models of infection. Using staining and biochemical techniques, we have characterized the cell wall of mar1∆ mutant cells, and by fluorescent microscopy we have demonstrated that the -(1,3)-glucan synthase catalytic subunit, Fks1, is mislocalized in mar1∆ cells. Using in vitro co-culture models, we have determined that the mar1∆ cell wall induces increased macrophage activation that is dependent on the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR-2 pattern recognition receptors.
To further understand the impact of the Mar1 protein on the host-pathogen interaction, we used in vivo mouse models to characterize the pathogenesis and immune response to this strain. Using histopathology and light microscopy, we have shown that mar1∆ cells induce granulomas in the lungs of infected mice, an in in vitro co-culture models we have demonstrated that the mar1∆ strain induces increased markers angiogenesis. Finally using immunization strategies, we show that the mar1∆ strain does not induce a protective response against a secondary lethal challenge.
Lastly, using bioinformatics tools and batch sampling, we developed a novel computational tool to more efficiently analyze mutants of interest generated by forward genetic screens. We demonstrate the efficacy of this tool through proof of principle experiments that led us to the discovery of the Mar1 protein described above. Additional projects in our lab and others have already utilized this mutant analysis tool in C. neoformans and we propose that it can be ultimately applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.
Item Open Access Regulation of Morphogenetic Events in Saccharomyces cerevisiae(2018) Lai, Hung-HsuehTip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast, Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented towards that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single cell level and the role of polarisome during pheromone-induced polarized growth. We employed genetics and live cell microscopy to characterize cellular events. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin “priming” facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.
Item Open Access Structural Studies of Phospho-MurNAc-pentapeptide Translocase and Ternary Complex of a NaV C-Terminal Domain, a Fibroblast Growth Factor Homologous Factor, and Calmodulin(2013) Chung, ChihPinPhospho-MurNAc-pentapeptide translocase (MraY) is a conserved membrane-spanning enzyme involved in the biosynthesis of bacterial cell walls. MraY generates lipid I by transferring the phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl-phosphate. MraY is a primary target for antibiotic development because it is essential in peptidoglycan synthesis and targeted by 5 classes of natural product antibiotics. The structure of this enzyme will provide insight into the catalytic mechanism and a platform for future antibiotic development. MraY genes from 19 bacteria were cloned, expressed, purified and assayed for biochemical stability. After initial crystallization screening, I found that MraY from Aquifex aeolicus (MraYAA) produced diffracting crystals. Recombinant MraYAA is functional and shows inhibition by the natural inhibitor capuramycin. After extensive optimization of crystallization conditions, we extended the resolution limit of the crystal to 3.3 Å. The crystal structure, the first structure of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily, reveals the architecture of MraYAA and together with functional studies, allow us to identify the location of Mg2+ at the active site and the putative binding sites of both substrates. My crystallographic studies provide insights into the mechanism of how MraY attaches a building block of peptidoglycan to the carrier lipid.
Voltage-gated Na+ (NaV) channels initiate action potentials in neurons and cardiac myocytes. NaV channels are composed of a transmembrane domain responsible for voltage-dependent Na+ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins including members of the fibroblast growth factor homologous factor (FHF) family and calmodulin (CaM). Through the collaboration between our lab and Geoffrey Pitt's lab, we report the first crystal structure of the ternary complex of the human NaV1.5 CTD, FGF13, and Ca2+-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform to understand roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity between individual NaV CTD isoforms and distinctive FHFs.
Item Open Access Structural Studies on the Lipid Flippase MurJ(2018) Kuk, Alvin Chun YinThe biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates membrane transport of oligosaccharide precursors from their cytoplasmic site of synthesis to their site of assembly outside the cytoplasm. To address this problem, cells utilize transporters such as those of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily to flip lipid-linked oligosaccharides across the cytoplasmic membrane. The MOP superfamily member MurJ has been shown to be the flippase that transports the lipid-linked peptidoglycan precursor lipid II, but the lack of structural information has limited our mechanistic understanding of the MurJ transport cycle. We determined the first crystal structure of MurJ (MurJTA from Thermosipho africanus) to 2.0-Å resolution, which assumed an inward-facing conformation unlike all other outward-facing structures of MOP transporters. Our structural and mutagenesis studies provide insight into a putative model of lipid II binding and an alternating-access mechanism of transport.