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Updated: Jul 7, 2026
High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Published on: August 11, 2014
Identification of Salmonella SPI-2 secretion system components required for SpvB-mediated cytotoxicity in macrophages
Sara H Browne1, Patricia Hasegawa, Sharon Okamoto
1Department of Medicine 0640, School of Medicine, University of California at San Diego, La Jolla, CA 92093-0640, USA. shbrowne@ucsd.edu
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.
Insights
Salmonella
Area of Science:
- Microbiology
- Molecular Biology
- Cell Biology
Background:
- Salmonella Typhimurium utilizes type III secretion systems (TSS) to inject effector proteins into host cells.
- The Salmonella Pathogenicity Island 2 (SPI-2) TSS is crucial for intracellular survival and virulence.
- SpvB is an effector protein with ADP-ribosyl transferase activity, targeting host actin.
Purpose of the Study:
- To investigate the mechanism of Salmonella SpvB-mediated actin depolymerization.
- To determine the role of SPI-2 TSS components in SpvB effector function.
- To assess the contribution of SpvB and SifA to Salmonella virulence in vivo.
Main Methods:
- In vitro infection of human macrophages with Salmonella mutants.
- Analysis of actin filament integrity and caspase-3 activation.
- Competitive infection assays in mice to evaluate bacterial virulence.
Main Results:
- SpvB targets monomeric actin, causing F-actin depolymerization in macrophages.
- SPI-2 TSS components (ssaV, spiC, sseB, sseC, sseD) are essential for SpvB-mediated actin disruption.
- SpvB-induced cytotoxicity and caspase-3 activation depend on SPI-2 TSS function.
- SpvB and SifA contribute independently to Salmonella virulence in a mouse model.
Conclusions:
- Salmonella SpvB disrupts host cell actin cytoskeleton via ADP-ribosyl transferase activity.
- The SPI-2 TSS is required for the delivery and function of SpvB in infected macrophages.
- SpvB and SifA are distinct virulence factors contributing to Salmonella pathogenesis.
