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

Author

• Rebekah Frances Hare

Date of Award

5-17-2014

Document Type

Dissertation

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.

Recommended Citation

Hare, Rebekah Frances, "Localization of Francisella pathogenicity island-encoded secreted proteins and their secretion system" (2014). Biological Sciences. 22.
https://ualaska.researchcommons.org/uaf_grad_bio_sciences/22

Handle

http://hdl.handle.net/11122/4541