In vivo genetic analysis indicates that PhoP-PhoQ and the Salmonella pathogenicity island 2 type III secretion system contribute independently to Salmonella enterica serovar Typhimurium virulence.

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium. Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Identification and analysis of bacterial virulence genes in vivo.

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

The effect of beta-blockade on plasma potassium concentrations and muscle excitability following static exercise.

The effects of beta-blockade on plasma [K+], muscle excitability and force during fatiguing exercise were examined. Nine healthy males (mean age 22.3+/-1.7 yr) performed a 3-min fatigue protocol that consisted of a sustained submaximal contraction (30% of the maximal voluntary contraction, MVC) of the right quadriceps muscle. Subjects performed the exercise after treatment with either placebo, beta1-selective (metoprolol, 100 mg) or an equipotent dose of non-selective beta1,2-blockade (propranolol, 80 mg, n=6; 100 mg, n=2; 120 mg, n=1) twice daily for 3 days before testing according to a randomized double-blind design. Brachial arterial and femoral venous blood samples were drawn before, during, and for 15 min following the contraction, together with maximal stimulation of the right femoral nerve to evoke a twitch and a compound muscle action potential (M-wave); the M-wave amplitude being used as an index of sarcolemmal excitability. The exercise-induced rise in plasma [K+] did not differ between treatments, but K+ re-uptake during recovery was slower following propranolol. The recovery of the twitch was significantly related to the recovery of plasma [K+] in all trials, but the evoked M-waves were unaffected by either the contraction or the drug treatment. Propranolol resulted in a significantly (P<0.05) greater reduction (51.9+/-7.3%) in MVC following the 3-min contraction compared with metoprolol (40.7+/-3.6%) or placebo (38. 9+/-3.6%). These results suggest that while beta1,2-blockade may significantly affect the recovery of muscle force and K+ homeostasis after fatiguing exercise (presumably through an inhibition of the Na+,K+-ATPase), it does not appear to affect surface membrane excitability.