Date of Award

4-11-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology & Immunology

First Advisor

Matthew L. Nilles

Abstract

Yersinia pestis, the etiological agent of the plague, immobilizes the host's innate immune response by translocating proteins called Yops (Yersinia outer proteins) into the eukaryotic cytoplasm using a type III secretion system (T3SS) injectisome known as the Yersinia secretion complex (Ysc). The T3SS injectisome, in many ways, resembles a hypodermic needle, composed of oligomeric rings with distinct areas responsible for different functions and many of these areas share homology to the flagellar T3SS basal body inner and outer membrane rings. Starting with the cytoplasmic region of the inner membrane known as the C-ring, this is an area where secretion is energized, chaperones are removed from secreted proteins and regulation occurs. The periplasmic surface of the inner membrane is composed of an oligomeric ring known as the MS ring that links to the outer membrane ring by a short rod through the cell wall. This rod is the base structure where the '1355 needle attaches, the needle is a hollow tube composed of a small protein that polymerizes in a helical pattern and extends into the extracellular space. This needle is capped with a hydrophilic protein that interacts with hydrophobic proteins secreted by the T3SS that oligomerize into the eukaryotic membrane forming a translocation pore. The translocation of effectors into the eukaryotic cytoplasm can then commence with certain effectors causing a feedback inhibition on translocation by dosing the translocation pore. The purpose of this work is to understand how the needle cap protein LcrV interacts with the translocation pore and how Ysc components bind to LcrG to negatively regulate secretion and which Ysc components are involved in disrupting the LcrG-Ysc complex. Disruption of charged extracellular surface exposed residues of the LcrV protein using hydrophobic alanines was monitored by observing changes in translocation. Two surface exposed areas near the C-terminus of the α7 helix and an undefined loop in LcrV between the β6 strand and the α12 helix were found to be important for translocation of Yop effectors into the eukaryotic cell. The determination of which Ysc component of the T3SS binds to LcrG suggests a Ysc conformation change or multiple Ysc's may be necessary for LcrG complex formation. YscD, a proposed component of the MS ring and YscL, a component of the C-ring, may disrupt the LcrG complexes. These observations suggest that Ysc apparatus binding by LcrG occurs at a Ysc near one of the inner membrane ring structures and both YscD and YscL may be important in disrupting this interaction to allow secretion.

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