Analysis of effectors of the Salmonella Typhimurium SPI‐2 type three secretion system

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium), an intracellular pathogen, causes gastroenteritis in humans and a systemic disease in mice. The ability of Salmonella to replicate inside host cells requires translocation of effector proteins across the vacuolar membrane, mediated by the Salmonella pathogenicity island‐2 (SPI‐2) type three secretion system (T3SS). However, the repertoire of effectors involved in this process has not been defined. The first part of this PhD work focused on SrfJ, a putative effector of the SPI‐2 T3SS with similarity to human lysosomal glucosylceramidase. Expression of its gene was dependent on SsrA/B, a two‐component regulatory system required for expression of most SPI‐2 effector genes. Expression of srfJ was also shown to occur under SPI‐2 T3SS activation conditions. However, there was no detectable secretion or translocation of the protein, although a srfJ mutant strain had an intracellular replication defect in primary bone marrow‐derived macrophages. Using a dual‐fluorescence reporter system that allows direct measurement of intracellular replication, the contribution to replication of individual SPI‐2 T3SS effectors was investigated. The replication kinetics of S. Typhimurium deletion mutants for all known SPI‐2 effectors were measured and compared in mouse bone marrow‐derived macrophages. Several mutant strains with replication defects were identified, thereby revealing that intracellular replication is the result of the contribution of numerous effectors. Two S. Typhimurium polymutant strains were generated whose replication defects closely resemble that of a SPI‐2 T3SS null mutant and are severely attenuated in virulence in vivo. These strains retained an intact T3SS and delivered a CD8+ T cell epitope via the SPI‐2 T3SS into the cytoplasm of infected cells. Since an S. Typhi mutant strain lacking the SPI‐2 T3SS has been shown to be safe and immunogenic in humans, these polymutant strains could have applications in vaccine design, as carrier strains for the delivery of heterologous antigens.