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The Salmonella SPI-2 Type III secretion system: regulation of a substrate specificity switch and functional analysis of the SpvD effector

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Title: The Salmonella SPI-2 Type III secretion system: regulation of a substrate specificity switch and functional analysis of the SpvD effector
Authors: Grabe, Grzegorz
Item Type: Thesis or dissertation
Abstract: One of the main determinants enabling Salmonella enterica serovar Typhimurium to replicate inside host cells is a type III secretion system (T3SS; an injectisome) encoded by Salmonella pathogenicity island 2 (SPI-2). This multi-protein structure assembles upon acidification of a Salmonella-containing vacuole (SCV) that surrounds Salmonella following host cell entry. It functions as a conduit that connects the bacterium to the host cytosol and translocates so called “SPI-2 effector” proteins that manipulate the host to support bacterial growth. The delivery of these proteins is spatiotemporally controlled by a gatekeeper complex composed of SsaL/SsaM/SpiC proteins (Yu et al., 2010). The gatekeeper prevents effector protein translocation, but is required for the export of translocon proteins that enable effectors to cross the vacuolar membrane into the cell. The SPI-2 effector proteins are translocated to the host cell only when the gatekeeper dissociates away from the injectisome, a process initiated by sensing a low-to-neutral pH shift (Yu et al., 2010). The functions of the SPI-2 injectisome and gatekeeper can be studied by growth of Salmonella at low pH medium followed by a shift to pH 7.2 (Beuzón et al., 1999; Yu et al., 2010). In a detailed alanine scanning mutagenesis of SsaL it was demonstrated that several amino acid positions, mainly located at the C-terminal domain of SsaL, are critical for the gatekeeper function when tested in an in vitro secretion assay. One mode of action of the gatekeeper could rely on a specific interaction with the translocon proteins that enables their subsequent secretion. Interactions between SsaL and the translocon proteins SseB and SseC were detected, however their exact manner requires further investigation. In a second part of this work, I focused on biochemical analysis of SpvD, an anti-inflammatory effector protein of unknown function encoded by the spv operon within pSLT virulence plasmid (Rolhion et al., in preparation). During this study it was determined that SpvD acts in vitro as a protease and that this activity depends on cysteine C73. A crystal structure determined as a result of collaboration with Dr Stephen Hare clearly shows that SpvD adopts a papain-like fold with a C73/H162/D182 catalytic triad, a characteristic feature and key element required for the enzymatic activities of this superfamily of proteins. A detailed analysis of the protein structure revealed a putative inhibitory arginine R161 that might obstruct the activity of SpvD. A subsequent BLAST search of SpvD amino acid sequences demonstrated serovar-specific differences at position 161. Mutations of the Typhimurium SpvD variant that mimicked SpvD from other serovars increased the proteolytic activity in an in vitro cleavage assay. Whether this polymorphism reflects host adaptation or partial loss of activity requires further investigation, as does identification of the physiological substrate(s) of SpvD.
Content Version: Open Access
Issue Date: Sep-2015
Date Awarded: Feb-2016
URI: http://hdl.handle.net/10044/1/57035
DOI: https://doi.org/10.25560/57035
Supervisor: Holden, David
Yu, Xiujun
Sponsor/Funder: Wellcome Trust (London, England)
Funder's Grant Number: WMNM_PS1709
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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