Localization of Francisella pathogenicity island-encoded secreted proteins and their secretion system

Hare, Rebekah Frances

dc.contributor.author	Hare, Rebekah Frances
dc.date.accessioned	2014-10-25T00:29:39Z
dc.date.available	2014-10-25T00:29:39Z
dc.date.issued	2014-05
dc.identifier.uri	http://hdl.handle.net/11122/4541
dc.description	Dissertation (Ph.D.) University of Alaska Fairbanks, 2014
dc.description.abstract	Intracellular pathogens have evolved virulence genes that allow them to exploit host cells for their life cycles, and virulence genes are commonly located in pathogenicity islands, such as the Francisella pathogenicity island of Francisella tularensis. The Francisella pathogenicity island is linked to virulence, intracellular growth, and a type VI secretion system. Since the Francisella pathogenicity island encodes a secretion system, I hypothesize that Francisella pathogenicity island encoded proteins are secreted during infection of host cells. The molecular mechanisms involved in the pathogenesis of this bacterium are not well understood and there are no readily available tools for studying these mechanisms. Therefore, I developed expression plasmids of all Francisella pathogenicity island encoded proteins as C-terminal and N-terminal epitope FLAG-tagged proteins. The Francisella pathogenicity island encoded proteins expressed from these plasmids successfully restored the intramacrophage growth phenotype in mutants of their respective genes that were deficient for intramacrophage growth. Immuno-fluorescence microscopy experiments of cells infected with bacteria containing the expression plasmids showed some of the Francisella pathogenicity island encoded proteins were secreted. To test if protein localization is dependent on the type VI secretion system, localization observed in wild type was compared to the localization of Francisella pathogenicity island encoded proteins in a pdpB mutant, a gene that is homologous to a type VI secretion system structural inner membrane protein. The localization of FLAG-tagged proteins was significantly reduced when expressed in the pdpB mutant compared to expression in wild type. Two of the secreted proteins, pdpC and pdpE, were tested for their roles in pathogenicity. pdpC was required for virulence in vivo but not for growth within macrophages. Plasmid expression of PdpC-FLAG and FLAG-PdpC in the pdpC mutant restored the virulent phenotype to that of the wild type. PdpE was not required for intramacrophage growth or virulence in mice. These data further support the hypothesis that the Francisella pathogenicity island encodes a secretome that contributes to the virulence of Francisella.
dc.description.tableofcontents	Chapter 1: Introduction of Francisella tularensis Host Cell Interactions -- 1.1. Bacteria-Host Cell Interactions -- 1.2. Phagocytosis and Phagosomal Maturation -- 1.2.1. Avoiding Phagosomal Destruction -- 1.3. Pathogenicity Islands -- 1.4. Secretion Systems -- 1.5. Francisella tularensis -- 1.5.1. Francisella Disease and Treatment -- 1.5.2. Epidemiology -- 1.6. Francisella Intracellular Life Cycle -- 1.7. The Francisella Pathogenicity Island -- 1.8. Summary -- 1.9. References -- Chapter 2: Development of Genetic Tools for Studying the FPI -- 2.1. Abstract -- 2.2. Introduction -- 2.3. Materials and Methods -- 2.3.1. Bacterial Cultures -- 2.3.2. DNA Manipulations -- 2.3.3. Transforming E. coli -- 2.3.4. Transforming Francisella -- 2.3.5. SDS-PAGE and Western Blotting -- 2.3.6. Macrophage Growth Assay -- 2.4. Results and Discussion -- 2.4.1. Development of the Francisella Expression Plasmids -- 2.4.2. C-terminus FLAG-tagged FPI Protein Expression in E. coli -- 2.4.3. FLAG-tagged FPI Protein Expression in F. novicida -- 2.4.4. Intramacrophage Growth Complementation -- 2.5. Acknowledgments -- 2.6. References -- 2.7. Chapter 2 Tables and Figures Legends -- 2.7.1. Table 2.1 Bacterial Strains Used in This Study -- 2.7.2. Table 2.2 Primers Used in This Study -- 2.7.3. Table 2.3 Plasmids Used in This Study -- 2.7.4. Figure 2.1 Diagram of FPI in F. novicida -- 2.7.5. Figure 2.2 Experimental Outline -- 2.7.6. Figure 2.3 Francisella Expression Plasmids -- 2.7.7. Figure 2.4 C-terminal FLAG-tagged Protein Expression in E. coli -- 2.7.8. Figure 2.5 FPI FLAG-tagged Protein Expression in F. novicida -- 2.7.9. Figure 2.6 Intracellular Growth -- Chapter 2 Tables -- Chapter 2 Figures -- Chapter 3: Localization of Francisella Pathogenicity Island Encoded Proteins within Infected Macrophage-like Cells -- 3.1. Abstract -- 3.2. Introduction -- 3.3. Materials and Methods -- 3.3.1. Bacterial and Cell Cultures -- 3.3.2. SDS-PAGE and Western Blotting -- 3.3.3. Immuno-fluorescence Microscopy -- 3.4. Results -- 3.4.1. Bacteria Co-Localization with C-terminal FLAG-tagged FPI Proteins During Cell Infection -- 3.4.2. C-terminal FLAG-tagged FPI Protein Localization within infected cells -- 3.4.3. Bacteria Co-Localization with N-terminal FLAG-tagged FPI Proteins During Cell Infection -- 3.4.4. N-terminal FLAG-tagged FPI Protein Localization within infected cells -- 3.4.5. Localization of FPI-Encoded Proteins within Infected Cells -- 3.4.6. Localization is Dependent on PdpB -- 3.5. Discussion -- 3.6. Authors' Contributions -- 3.7. Acknowledgements -- 3.8. References -- 3.9. Chapter 3 Figure Legends -- 3.9.1. Figure 3.1 Bacteria Associated with FPI C-tagged Proteins -- 3.9.2. Figure 3.2 Infected Macrophages with FPI C-tagged Proteins -- 3.9.3. Figure 3.3 Bacteria Associated with FPI N-tagged Proteins -- 3.9.4. Figure 3.4 Infected Macrophages with FPI N-tagged Proteins -- 3.9.5. Figure 3.5 Localization of FPI Proteins within Infected macrophages -- 3.9.6! Figure 3.6 Localization of FPI Proteins During Infection of Sua-1B Cells -- 3.9.7. Figure 3.7 IglC Secretion is Dependent on T6SS -- 3.9.8!. S. Figure 3.1 Three-dimensional Reconstruction of IglA -- 3.9.9. S. Figure 3.2 Three-dimensional Reconstruction of IglI -- 3.9.10. S. Figure 3.3 Three-dimensional Reconstruction of PdpE -- Chapter 3. Tables -- Chapter 3. Figures -- Chapter 4: PdpC is a Secreted Protein of Francisella novicida -- 4.2. Introduction -- 4.3. Materials and Methods -- 4.3.1. Bacterial Strains and Plasmids -- 4.3.2. Polymerase Chain Reaction (PCR) and Primer Design -- 4.3.3. Subcellular Localization of PdpC -- 4.3.4. Targeted Integration of FLAG Tag into the F. novicida chromosome -- 4.3.5. Chemical Transformation of F. novicida -- 4.3.6. SDS-PAGE and Western Blotting -- 4.3.7. Mouse Infections -- 4.3.8. Chicken Embryo Infections -- 4.3.9. Immuno-fluorescence Microscopy -- 4.4. Results -- 4.4.1. PdpC Expression in F. novicida -- 4.4.2. The Roles of PdpC in Virulence of Mice -- 4.4.3. pdpC is Required for Virulence of F. novicida in Embryonated chicken eggs -- 4.4.4. The Role of PdpE in Virulence -- 4.4.5. PdpC is Localized to the Host Cells During Infection -- 4.4.6. PdpC Localization During Infection is Dependent on T6SS -- 4.4.7. Localization of PdpC-FLAG to Host Cells Does Not Depend on Overexpression of PdpC-FLAG -- 4.4.8. Temporal Regulation of PdpC Secretion -- 4.4.9. PdpE is Secreted in a T6SS Dependent Manner -- 4.5. Discussion -- 4.6. Conclusions -- 4.7. Authors' Contributions -- 4.8. Acknowledgements -- 4.9. References -- 4.10. Chapter 4 Figure Legends -- 4.10.1. Figure 4.1 Expression of PdpC -- 4.10.2. Figure 4.2 Survival of Mice Infected Intradermally with F. novicida and ΔpdpC Mutant -- 4.10.3. Figure 4.3 Survival of Embryos with ΔpdpC Mutants of F. novicida -- 4.10.4. Figure 4.4 Phenotype of pdpE Mutant Francisella novicida -- 4.10.5. Figure 4.5 Detection of FLAG-tagged PdpC in Infected Host cells -- 4.10.6. Figure 4.6 PdpC-FLAG Localization is Dependent on T6SS -- 4.10.7. Figure 4.7 PdpC-FLAG Expressed From Bacterial Chromosome -- 4.10.8. Figure 4.8 Time Course of pdpC Detection in Infected Host cells -- 4.10.9. Figure 4.9 PdpE-FLAG Expressed in Wild Type or pdpB Mutant background -- 4.10.10. S. Figure 4.1 Three-dimensional Reconstruction of PdpC-FLAG in Infected Cells -- 4.10.11. S. Figure 4.2 Three-dimensional Reconstruction of PdpC-FLAG in infected cells -- Chapter 4. Tables -- Chapter 4. Figures -- Chapter 5: The Secretome within the FPI: Conclusions and Future Studies -- 5.1. References -- 5.2. Chapter 5. Figure Legend -- 5.2.1. Figure 5.1 Model of Francisella's T6SS -- Chapter 5 Figures.
dc.title	Localization of Francisella pathogenicity island-encoded secreted proteins and their secretion system
dc.type	Dissertation
dc.type.degree	phd
dc.identifier.department	Department of Biology and Wildlife
dc.contributor.chair	Hueffer, Karsten
dc.contributor.committee	Taylor, Barbara
dc.contributor.committee	Duffy, Larry
refterms.dateFOA	2020-03-05T09:28:08Z