Pathogenicity of Salmonella: SopE-mediated membrane ruffling is independent of inositol phosphate signals

Abstract

Studies [Zhou, D., Chen, L.-M., Hernandez, L., Shears, S.B., and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248-259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen's ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis.

Fig. 1

Reactivity of SopB and SopE towards InsP₅ in vitro. Recombinant GST-SopB (0.12 mg/ml final concentration) or GSTSopE (1.1 mg/ml final concentration) was incubated at 30 °C for 0– 40 min in 25 μl of assay buffer containing 50 mM HEPES (pH 7.2), 10 mM DTT, 0.35 mg/ml bovine serum albumin. Assays were quenched and neutralized as described in Section 2 and the InsP₄ product was separated from the InsP₅ substrate by batch ion-exchange chromatography [26]. Data shown are triplicates from a representative experiment that was repeated four times with identical results. In other experiments (not shown) SopE also did not dephosphorylate InsP₅ when the assay buffer was supplemented with 2 mM MgSO₄.

Fig. 2

Transient expression of SopE in PTEN-null glioblastomas induces the formation of discrete patches of membrane ruffling. Human PTENnull U87MG glioblastoma cells were either mock-transfected (‘‘control’’, left panel), or SopE-transfected (middle and right panels). After 24 h, cells were labeled with Texas Red-X Phalloidin, to identify actin. The yellow arrows highlight the discrete patches of actin that signify membrane ruffling. In the right-hand panel the green colour shows GFP fluorescence in cells in the same field of view as the middle panel. The white scale bar in the GFP image represents 20 μm.

Fig. 3

SopE transfection of PTEN-null glioblastomas does not affect cellular levels of inositol phosphate signals. Glioblastoma cells were radiolabeled with [³H]inositol as described in Section 2. Cells were either mock-transfected (“control”) or transfected with either SopB, or SopE and GFP, for either 6, 12 or 24 h, and then cellular inositol phosphate levels were determined by HPLC. Two representative HPLC profiles are shown for cells after 24 h. transfection (Panels A and B). Panel C shows levels of InsP₅ at various times after transfection with SopE; data are means and standard errors from 2–4 experiments at each time point. Panel D shows the levels of InsP₅, 24 h after transfection with SopB; data are means and standard errors from three experiments. *P < 0.002.

Fig. 4

Induction of PTEN expression in the glioblastoma cells PTEN-null U87MG glioblastoma cells were treated with either 0.5 μM muristerone for 48 h or an appropriate volume of the vehicle control. Cell lysates were prepared and 40 μg of total protein was resolved on a gel and immunoblotted with antibodies specific for PTEN or GAPDH.

Fig. 5

Inositol phosphates and membrane ruffling in SopE-transfected glioblastoma cells in which PTEN is expressed. Cells in which PTEN expression had been induced by 48 h treatment with 0.5 μM muristerone (see Fig. 4) were either mock-transfected (“control”) or transfected with SopE and GFP for a further 24 h. Cells were labeled with Texas Red-X Phalloidin, to identify actin. Some muristerone-treated cell cultures were also radiolabeled with [³H]inositol for 3 days prior to transfection (control or SopE) and then inositol phosphate levels were ascertained by HPLC analysis. The white scale bar in the GFP image represents 20 μm.