| Summary: | In causing gastroenteritis, Salmonella typhimurium are able to invade and grow within
non-phagocytic intestinal cells. They are also able to survive within phagocytic cells, and have
the potential to cause systemic disease. The ability of these bacteria to be intracellular is
important in the disease process.
Salmonella express an unique set of genes inside macrophages (not expressed with
other stresses, e.g. heat, low pH, starvation), however, only a few have been identified.
Therefore, the goal of this study was to search for Salmonella genes only expressed inside
cells, thereby identifying genes essential for intracellular survival and perhaps virulence.
A light-based reporter system was developed to specifically detect gene expression
from intra-cellular bacteria. Vibrio harveyi luciferase genes, luxAB, were fused to Salmonella
plasmid virulence genes, spvRAB, and the construct was shown to be upregulated after
Salmonella invasion into epithelial cells. Upregulation was also demonstrated using a similar
B-galactosidase (lacZ) reporter gene fusion. The results indicated luciferase was a sensitive
reporter, able to monitor bacterial gene expression from within host cells, and able to
differentiate live from dead bacteria.
Subsequently, a library of S. typhimurium mutants was made by randomly inserting
promoterless luxAB genes into the bacterial chromosome. Individual mutants were screened
for luciferase expression (i.e. light production) within phagocytic cells. Extracellular bacterial
mutants expressing light during growth in rich media or in the presence of cell-secreted factors
were eliminated.
From this screen, four S. typhimurium genes were identified as upregulated within
both phagocytic and non-phagocytic cells. Three genes were found located within "Salmonella
pathogenicity islands" (SPI): sigD/sopB and pipBwithin SPI-5 and ssaR within SPI-2. The
sigD/sopB gene encodes an inositol phosphate phosphatase affecting host cell signalling
pathways and chloride secretion. The ssaR gene potentially encodes a type III secretion
apparatus component. Bacterial type III secretion systems are highly regulated and specialized
for secretion of bacterial proteins directly into host cells. The fourth gene (iicA for induced
mtracellularly) was completely novel. The four bacterial mutants retained their ability to invade
and grow within cultures cells, and all but iicA were requiredfor virulence in a mouse model.
This work confirms Salmonella pathogenicity includes genes expressed in the
intracellular environment. The ability to identify such genes, and their further characterization
has led to an enhanced understanding about how Salmonella functions as an intracellular
pathogen, in addition to identifying specific virulence factors. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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