Structural and functional analysis of the Salmonella pathogenicity island 4 encoded type-I secretion system and its interaction with a methyl-accepting chemotaxis protein

Please use this identifier to cite or link to this item:
Open Access logo originally created by the Public Library of Science (PLoS)
Title: Structural and functional analysis of the Salmonella pathogenicity island 4 encoded type-I secretion system and its interaction with a methyl-accepting chemotaxis protein
Authors: Hoffmann, Stefanie
Thesis advisor: Prof. Dr. Michael Hensel
Thesis referee: Prof. Dr. Guntram Grassl
Abstract: Salmonella Typhimurium is a Gram-negative, facultative intracellular and anaerobic bacterium with high importance as an intestinal pathogen. For successfully colonization and persistence within the host S. Typhimurium utilizes a set of virulence-associated secretion systems. One of these systems is the type I secretion system (T1SS) encoded by Salmonella pathogenicity island 4 (SPI-4). SPI-4 encodes for an adhesin which is necessary to establish initial contact between the bacteria and host cells. Subsequently, the type 3 secretion system encoded by SPI-1 (T3SS-1) injects its effector proteins into the host cell leading to the internalization of the bacteria. SPI-4 harbors six genes, siiAF. SiiCDF form the canonical subunits of the T1SS, SiiE is the only known substrate and the two accessory proteins SiiAB probably form a proton channel. SiiE is an approximately 600 kDa large secreted adhesion. Interestingly, there is only a temporal stable retention of SiiE at the bacterial surface where it can exert its adhesin function. SiiAB is thought to play an important role in the switch between secretion and stable retention of SiiE as their deletion leads to reduced adhesion and invasion of polarized epithelial cells while secretion is only mildly affected.The aim of this thesis was to further characterize the role of SiiAB for SiiE-mediated adhesion and to identify possible environmental signals for the switch between retention and secretion of SiiE. Invasion of polarized epithelial cells was used as a main readout to quantify SPI-4 function. For that the “Virtual Colony Count” method was established as an alternative analytical method and compared to the classical counting of colony-forming units (CFUs). With this automated evaluation method it was possible to increase the throughput of infection experiments and to minimize errors by manual CFU counting. SiiAB has sequence and structural homologies to MotAB and other ion- conducting channels such as PomAB, ExbBD or TolQR, which is why a similar function is assumed. By ratiometric pH measurements in living S. Typhimurium cells it could be shown that overexpression of the supposed proton channel SiiAB actually leads to a drop in intracellular pH. The mutation of a conserved aspartate residue at position 13 of SiiA resulted in loss of proton-conducting function of the SiiAB complex. The same holds true for aspartate 32 of MotB, which could confirm the hypothesis of the functionality of SiiAB. In the present study, the methyl-accepting chemotaxis protein CheM was identified and characterized as an interaction partner of SiiAB. It could be shown that the perception of aspartate by CheM shifted the ratio of secreted and retained SiiE towards the retention of the adhesin. While this effect was independent of other motility or chemotaxis components, the transduction of this signal is probably mediated through the interaction with SiiAB. The stimulation of CheM thus has a direct influence on the SPI-4 mediated adhesion of the bacteria and thus represents an example of a noncanonical signal transduction of a MCP to a virulence factor. It remains to be clarified whether this interaction is also important in vivo for a successful infection of Salmonella. Here, components of the mucus layer could provide the signals for the CheM sensor molecule resulting in precise triggering of SiiE-mediated adhesion in the enterocyte vicinity.
Subject Keywords: Salmonella
Issue Date: 10-May-2022
Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

Files in This Item:
File Description SizeFormat 
thesis_hoffmann.pdfPräsentationsformat6,56 MBAdobe PDF

Items in osnaDocs repository are protected by copyright, with all rights reserved, unless otherwise indicated.