Date of Award

4-3-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology & Immunology

First Advisor

Matthew Nilles

Abstract

Yersinia pestis, the causative agent of plague, is a highly pathogenic gram negative bacterium that attenuates the host immune response by translocating effector proteins, known as Yops, directly from the bacterial cytoplasm into the eukaryotic cell cytoplasm using a Type III secretion system. The Type III secretion system uses a polymerized needle to translocate Yops into the eukaryotic cell. This self-polymerizing needle is composed of YscF. YscF has been tested as a possible vaccine alternative to other subunit vaccines. Wild type YscF has been previously shown to provide a low amount of protection against challenge with Y. pestis. The focus of this work was to improve YscF as a vaccine candidate. Major epitopes in YscF were mapped to the N-terminus and C-terminus of the protein. The major epitope of YscF was predicted to lie within the fully polymerized needle, obscured from view at the tip of the needle by LcrV, suggesting that the major epitope of YscF is not surface accessible to antibodies, a possible reason for the low level of protection seen in YscF immunization studies. One of the minor epitopes is accessible to antibodies on the surface of the polymerized needle. To drive the antibody response towards the minor epitope, a YscF mutant missing its N-terminus was analyzed. Truncated YscF (trYscF) is not protective. High proinflammatory cytokine levels, were detected in pre-infection sera from mice vaccinated with trYscF. Cytokine levels from trYscF-vaccinated mice were higher than in pre-infection mouse sera from wtYscF vaccinated mice. YscF may have a concealed pathogen associated marker (PAMP) that is exposed in trYscF. Monocytes produced significantly higher amounts of proinflammatory cytokines when treated with trYscF compared to cells treated with wild type YscF. Macrophages produced a significantly higher amount of TNF-α when stimulated with trYscF compared to wild type YscF. NF-κB was found to be activated in cells treated with trYscF and wt YscF. Results demonstrate that YscF represents a new class of pathogen associated molecular patterns.

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