Date of Graduation

8-2014

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Danielle A. Garsin

Committee Member

Joseph Alcorn

Committee Member

Steve Norris

Committee Member

Swathi Arur

Committee Member

Ambro van Hoof

Abstract

The production of ROS (reactive oxygen species) in response to pathogen detection is a rapid, nonspecific response that is evolutionarily conserved from nematodes to humans. ROS serve as direct and indirect effectors of innate and adaptive immunity. In Caenorhabditis elegans, a ROS burst is observed during infection and is mediated by the dual oxidase BLI-3, which produces H2O2. RNAi (RNA interference) to reduce the amount of BLI-3 results in a significant increase in susceptibility to pathogens, suggesting BLI-3 has a role in the immune response. However, H2O2 by itself is not a potent antimicrobial and in other systems is converted to a more potent oxidant by an affiliated peroxidase. During my work, I have characterized a group of previously unstudied peroxidases in C. elegans and determined their involvement in the host immune response to Enterococcus faecalis. In particular, I focused on SKPO-1 (ShkT-containing peroxidase) and how it contributes to the host immune response with respect to BLI-3.

By RNAi and skpo-1 mutant analysis, I determined that SKPO-1 is involved in the host immune response during E. faecalis infection. By tissue-specific RNAi, I determined that SKPO-1 is functionally active in the hypodermis and required for wild type resistance to infection. Additionally, by immunohistochemistry, I observed that SKPO-1 is only expressed in the hypodermis and that its protein levels do not change in response to E. faecalis. In support of SKPO-1 acting as a peroxidase, I observed a significant increase in H2O2 levels when expression of the gene was reduced by RNAi. The increased H2O2 was observed only during infection and was BLI-3-dependent. Thus, I have characterized a likely BLI-3/SKPO-1 system, potentially similar to the oxidative burst systems present in higher eukaryotes.

Keywords

Peroxidase, SKPO-1, ROS, innate immunity, E. faecalis, C. elegans, hypodermis

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