The putative thiosulfate sulfurtransferases PspE and GlpE contribute to virulence of <em>Salmonella</em> Typhimurium in the mouse model of systemic disease.

The phage-shock protein PspE and GlpE of the glycerol 3-phosphate regulon of Salmonella enterica serovar Typhimurium are predicted to belong to the class of thiosulfate sulfurtransferases, enzymes that traffic sulfur between molecules. In the present study we demonstrated that the two genes contribute to S. Typhimurium virulence, as a glpE and pspE double deletion strain showed significantly decreased virulence in a mouse model of systemic infection. However, challenge of cultured epithelial cells and macrophages did not reveal any virulence-associated phenotypes. We hypothesized that their contribution to virulence could be in sulfur metabolism or by contributing to resistance to nitric oxide, oxidative stress, or cyanide detoxification. In vitro studies demonstrated that glpE but not pspE was important for resistance to H(2)O(2). Since the double mutant, which was the one affected in virulence, was not affected in this assay, we concluded that resistance to oxidative stress and the virulence phenotype was most likely not linked. The two genes did not contribute to nitric oxid stress, to synthesis of essential sulfur containing amino acids, nor to detoxification of cyanide. Currently, the precise mechanism by which they contribute to virulence remains elusive

Survival of wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains in the presence of peroxynitrite treatment. Bacteria grown to logarithmic phase (OD_600nm = 0.04±0.01) were treated with 360 µM peroxynitrite for 15 min.

<p>Survival of bacteria was determined by calculating the number of bacteria after 15 min in relation to the number of bacteria at the beginning of the experiment. Results show mean values of at least three independent experiments ± SEM.</p

Competitive indices of <i>S</i>. Typhimurium 4/74 wild type bacteria relative to mutant bacteria in the mouse spleen.

<p>C57/BL6 mice were infected i.p. with equal numbers of mutant and wild type bacteria (each 5×10³ CFU). After 4 to 6 days, mice were sacrificed and the spleen was removed. Serial dilutions were spotted on LB agar plates and number of wild type and mutant bacteria in a total of 100 colonies was further determined by selection of the resistance marker. Competitive indices (C.I.) were calculated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070829#pone.0070829-Jelsbak1" target="_blank">[42]</a>. The results are shown as mean values ± STD based on the number of mice tested as indicated in brackets. Significant differences from 1.0 (**p<0.01) were determined by one-sample t-test analysis.</p

Growth of WT (4/74) and <i>pspE</i> and <i>glpE</i> mutated and complemented strains in M9 medium with 10 mM H₂O₂.

<p>Wild type (black), Δ<i>glpE</i> (blue), Δ<i>pspE</i> (green), Δ<i>glpE</i>/Δ<i>pspE</i> (red) and Δ<i>glpE</i>+<i>glpE</i> strains were inoculated to an OD₆₀₀ value of 0.05, H₂O₂ was added and growth was followed for 16 hours. Results shown are representative of two biological repeats.</p

Growth of wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains in the presence 0.01% SDS.

<p>Strains were grown in 100 ml flasks containing 20 ml M9 supplemented with 0.01% SDS. The growth of wild type and mutated strains was similar.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Infection of J774 macrophages with wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains.

<p>J774A.1 macrophages were infected with complemented-opsonized bacteria in a MOI of 10 and incubated for 25 min. Extracellular bacteria were killed by treatment with 100 µg ml⁻¹ gentamicin. Bacteria were released from the macrophages 1 h p.i., 4 h p.i. and 24 h p.i. and the number of intracellular bacteria was determined by CFU ml⁻¹ calculations and was expressed relatively to CFU at T1. The results show mean values ± SEM of at least three independent replicates. The small inserts shows results of an internal control experiment conducted with a SPI-2 (<i>ΔssaV</i>) deficient strain showing the expected reduced intracellular survival/replication of the mutated strain.</p