Functional dissection of translocon proteins of the Salmonella Pathogenicity Island 2-encoded type III secretion system

<p>Abstract</p> <p>Background</p> <p>Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. <it>Salmonella enterica </it>is a facultative intracellular pathogen that uses the <it>Salmonella </it>Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the <it>Salmonella</it>-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.</p> <p>Results</p> <p>Here we investigated the structural requirements of SseB and SseD to form a functional translocon. Based on bioinformatic predictions, deletional analyses of SseB and SseD were performed and the effect on secretion by the T3SS, formation of a translocon, translocation of effector proteins and intracellular replication was investigated. Our data showed that both SseB and SseD are very sensitive towards alterations of the primary structure of the proteins. Although proteins encoded by mutant alleles were still secreted, we observed that all mutations resulted in a loss of function of the SPI2-T3SS.</p> <p>Conclusion</p> <p>These observations indicate that translocon proteins of the SPI2-T3SS are highly evolved towards the formation of multi-subunit complex in the host cell membrane. Structural alterations are not tolerated and abrogate translocon function.</p

Divergent Roles of Salmonella Pathogenicity Island 2 and Metabolic Traits during Interaction of S. enterica Serovar Typhimurium with Host Cells

The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium (S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied

Intracellular proliferation of <i>S.</i> Typhimurium in various cell lines.

<p><i>S.</i> Typhimurium strains were used for infection of epithelial cell lines (A) or macrophage-like RAW 264.7 cells (B) at an MOI of 1 as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033220#pone-0033220-g001" target="_blank">Fig. 1</a>. Non-internalized bacteria were killed by the addition of gentamicin and infected host cells were lysed at 2 h or 16 h after infection in order to determine the number of intracellular bacteria. The x-fold intracellular replication is the ratio of intracellular bacteria at 16 h divided by the 2 h values. The graphs show means and standard deviations of assays performed in triplicate and data sets representative of three independent experiments are shown.</p

Bacterial strains and plasmids used in this study.

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

Growth of <i>S.</i> Typhimurium strains in cell culture media.

<p>A) <i>Salmonella</i> WT and various mutant strains were grown in DMEM cell culture medium without further additives. Cultures were grown in baffled flasks at 37°C with agitation at 180 rpm. Growth was recorded by determination of the OD₆₀₀. B) Effect of supplementation of cell culture media on growth of the <i>aroA</i>-deficient strain. <i>S.</i> Typhimurium WT (black symbols) and the <i>aroA</i> strain (green symbols) were grown in DMEM supplemented with 10 µg×ml⁻¹ 2,3- DHBA (triangles), 10 µg×ml⁻¹ pABA (squares) or both supplements (diamonds). C) Effect of supplementation of DMEM with 1 mM adenine on growth of WT and <i>purD</i>-deficient strains. Growth curves are representative for three independent replicates with similar growth characteristics.</p

Effect of auxotrophic mutations on the function of the SPI2-encoded T3SS in RAW264.7 cells.

<p>Murine macrophage-like cells were infected with <i>S.</i> Typhimurium WT or various mutant strains each harboring a plasmid for the expression of <i>sseJ</i>::HA at an MOI of 10. Detection of translocated SseJ-HA was detected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033220#pone-0033220-g006" target="_blank">Fig. 6</a>. Representative cells for each infecting strain are shown. Scale bars represent 10 µm.</p

Kinetics of invasion and intracellular proliferation of <i>S.</i> Typhimurium in MDCK cells.

<p>Polarized monolayers of MDCK cells were infected with various <i>S.</i> Typhimurium strains with an MOI of 1 as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033220#pone-0033220-g001" target="_blank"><b>Fig. 1</b></a>. A) The amount of bacteria in the inoculum (0 h) was determined by plating of serial dilutions of the inoculum onto agar plates. The amount of intracellular bacteria was determined at various time points after infection as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033220#pone-0033220-g002" target="_blank"><b>Fig. 2</b></a>. B) The proportion of internalized bacteria was determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033220#pone-0033220-g001" target="_blank"><b>Fig. 1</b></a>. C) The x-fold intracellular replication is the ratio of intracellular bacteria at 3, 5, 8 or 20 h divided by the 1 h values. The graphs show means and standard deviations of assays performed in triplicates.</p

Cell densities of <i>S.</i> Typhimurium WT and the <i>aroA</i>-deficient strain after 24 h growth in various media.

*<p>Supplements: pABA, DHBA, each at 10 µg×ml¹, F, phenylalanine, W, tryptophan, Y, tyrosine, each at 40 µg×ml¹, Casa, 0.1% casamino acids.</p>**<p>The OD₆₀₀ was recorded after 24 h growth of 3 ml cultures in test tubes with aeration by rotation in a roller drum at 37°C.</p

Internalization of <i>S.</i> Typhimurium by various cell lines.

<p>A) Invasive <i>S.</i> Typhimurium WT (black bars), and isogenic SPI2-deficient (<i>ssaV</i>, red bars) or auxotrophic Δ<i>purD</i> (green bars) and Δ<i>aroA</i> (yellow bars) strains were used to infect various host cell lines at an MOI of 1. HeLa and MDCK epithelial cells were grown in multiwell dishes. The human epithelial cell lines T84 and CaCo2 were either grown directly in multiwell dishes (w/o filter) or using transwell membrane filter inserts in order to allow differentiation to polarized monolayers (transwell). After invasion for 1 h and killing non-internalized bacteria by addition of gentamicin, the cells were lysed by addition of 0.1% Triton X-100 in PBS (HeLa, RAW) or 0.5% desoxycholate in PBS (MDCK, T84, CaCo2), and serial dilutions of lysates were plated onto agar plates for determination of the amount of internalized bacteria. The internalization is expressed as percentage of the bacterial inoculum applied for invasion. B) Internalization of <i>S.</i> Typhimurium by the murine macrophage-like cell line RAW264.7 was quantified as for A), except that bacteria from stationary cultures were used for infection at an MOI of 1. The graphs show means and standard deviations of assays performed in triplicate and data sets are representative of three independent experiments are shown. Statistical significances were calculated for mutant vs. WT strains and indicated as n.s., not significant; *, P<0.05; **, P<0.01; ***, P<0.001.</p