Functional analysis of type III secretion systems in Salmonella enterica
Year: 2010
Type: dissertation
Abstract: The Gram-negative bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of human gastroenteritis. The pathogen expresses three different Type Three Secretion Systems (T3SS) in order to establish a successful infection: the flagella assembly system essential for bacterial motility as well as the Salmonella pathogenicity island (SPI) 1- and 2-encoded T3SS acting as molecular syringes for injection of effector proteins into the host cell cytoplasm. The SPI1-encoded T3SS is required for active invasion of non-phagocytic cells in the human intestine while the SPI2-encoded T3SS allows survival as well as replication of the bacterium within host cells. Salmonella is protected against the innate immune response of the host by repetitive O-antigen (OAg) units of the lipopolysaccharide. Short (up to 15 repeat units), long (16-35 repeat units) and very long (more than 100 repeat units) OAg species are present on the surface of S. Typhimurium and the OAg length pattern is controlled by the regulator proteins WzzST and WzzfepE. In this study, the regulators were deleted and the effect of different OAg length on the function of the Salmonella T3SS was investigated. A luciferase-based translocation assay for the quantification of SPI2-encoded T3SS mediated effector translocation was established. By the use of this assay, it was shown that the OAg length is not critical for the function of the SPI2-encoded T3SS. The motility of the bacterium was also not affected by modifications of the OAg length. In contrast, truncation of the OAg length promoted invasion into polarised as well as non-polarised epithelial cells and increased effector translocation mediated by the SPI1-encoded T3SS. However, the short OAg form impaired the resistance of Salmonella against human complement and antimicrobial peptides. In summary, the data suggest that the trimodal OAg chain length distribution in Salmonella is optimised towards a balance between invasion of epithelial cells and resistance against antibacterial factors. In general, T3SS are composed of a basal body spanning the Gram-negative cell envelope, a needle-like extension of the basal body and a translocon pore connected to the needle and integrated into the host cell membrane. Another aspect of this study included the functional dissection of the putative translocon proteins SseB and SseD of the SPI2-encoded T3SS. Domains of SseB were identified that are involved in expression and secretion of this protein in vitro. However, mutagenesis of sseB and sseD had a dramatic effect on Salmonella intracellular replication and effector translocation leading to a loss of function of the SPI2-encoded T3SS. This work emphasises the highly sensitive nature of these structural proteins against alterations of their primary structure.
Author: Stefanie U. Hölzer
Language: English
Subfield: Microbiology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3213599225