1. Identification of Salmonella SPI-2 secretion system components required for SpvB-mediated cytotoxicity in macrophages and virulence in mice.
- Author
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Browne SH, Hasegawa P, Okamoto S, Fierer J, and Guiney DG
- Subjects
- Actins metabolism, Animals, Caspase 3 metabolism, Cells, Cultured, Gene Deletion, Humans, Membrane Transport Proteins genetics, Mice, Mice, Inbred BALB C, Mutagenesis, Insertional, Protein Transport, Salmonella Infections, Animal microbiology, Salmonella enterica genetics, Spleen microbiology, ADP Ribose Transferases metabolism, Macrophages microbiology, Membrane Transport Proteins metabolism, Salmonella enterica metabolism, Salmonella enterica pathogenicity, Virulence Factors metabolism
- Abstract
The Salmonella SpvB protein possesses ADP-ribosyl transferase activity. SpvB, acting as an intracellular toxin, covalently modifies monomeric actin, leading to loss of F-actin filaments in Salmonella-infected human macrophages. Using defined Salmonella mutants, different functional components of the SPI-2 type three secretion system (TTSS), ssaV, spiC, sseB, sseC, and sseD, were found to be required for SpvB-mediated actin depolymerization in human macrophages. Expression of SpvB protein in Salmonella was not affected by any of the SPI-2 mutants and the effects of these loci were not due to reduced numbers of intracellular bacteria. Interestingly, the major SPI-2 virulence effector, SifA, is not required for SpvB action. Further, caspase-3 activation is an additional marker of cytotoxicity in Salmonella-infected human macrophages. Caspase-3 activity depended on SpvB and SPI-2 TTSS function, but not on SifA. These human macrophage cell culture results were corroborated by virulence studies in mice. Using competitive infection of mice with mixed inocula of single and double mutants, spvBmut1 mutation did not have an effect independent of ssaJ mutation, essential for SPI-2 TTSS function. In contrast, competitive infection studies in mice confirmed that SpvB and SifA have independent virulence effects, as predicted by the macrophage studies.
- Published
- 2008
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