Pseudomonas aeruginosa ExoU, a toxin transported by the type III secretion system, kills Saccharomyces cerevisiae

Abstract

ExoU, a protein transported by the type III secretion system of Pseudomonas aeruginosa, is an important cytotoxin, though its mechanism of action is unclear. Here we show that the intracellular expression of ExoU is cytotoxic to Saccharomyces cerevisiae. Furthermore, internal amino- and carboxyl-terminal deletions confirmed that regions of ExoU previously shown to be essential for killing mammalian cells were also required for killing yeast cells. These findings indicate that S. cerevisiae is a useful model organism for the study of ExoU.

FIG. 1.

ExoU is toxic to yeast. Yeast strain INVSc1 transformed with pVector or pExoU was streaked onto inducing (SC-Gal) or repressing (SC-Glc) agar and incubated at 30°C for 3 days.

FIG. 2.

Microscopic examination of yeast expressing ExoU. INVSc1(pVector) and INVSc1(pExoU) were grown under repressing conditions for 18 h, transferred to either repressing SC-Glc or inducing SC-Gal medium, and stained with Calcofluor and FUN-1. In metabolically active yeast cells, FUN-1 is cleaved and fluoresces red within vacuolar structures, while dead but intact yeast cells have a diffuse green fluorescence. Calcofluor causes yeast cell walls to fluoresce blue. The blue shells seen when INVSc1(pExoU) was grown under inducing conditions likely represent dead yeast cells that have extruded their cellular contents.

FIG. 3.

Minimal amounts of ExoU are sufficient for lethality. ExoU expression was controlled by varying the concentration of repressing sugar (glucose) or inducing sugar (galactose) in agar plates. Total sugar was kept constant at 2% by supplementing with raffinose, which does not affect GAL1 promoter activity. INVSc1(pVector) and INVSc1(pExoU) were grown under repressing conditions for 18 h, diluted to an OD₆₀₀ of 1.0, and inoculated onto agar plates. Colonies were counted after incubation at 30°C for 4 days. Note that the small number of viable colonies observed under inducing conditions is likely due to spontaneous suppressor mutations. Data were pooled from two separate experiments, each of which was done in triplicate.

FIG. 4.

Mutational analysis of ExoU. Wild-type and mutated forms of ExoU containing amino-terminal His and Xpress epitope tags were expressed in yeast cells and their cytotoxic activities were determined by streaking the yeast cells onto inducing (SC-Gal) agar. Deleted segments of the protein are indicated, as are the restriction endonucleases that were used to generate the deletions. To the right, the viability of yeast transformed with the mutated exoU alleles and grown on inducing agar is indicated. The C1 mutant had a partially cytotoxic phenotype.

FIG. 5.

Immunoblot analysis of yeast transformed with mutated exoU alleles. Yeast cells were grown under inducing conditions, diluted to 10⁷ CFU/ml, and allowed to grow for an additional 4 h. Yeast cells were lysed with glass beads and 20% trichloroacetic acid. Proteins from cell lysates were collected, electrophoresed through a sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane, and exposed to polyclonal ExoU antiserum. Only mutated forms of ExoU that lacked cytotoxic activity are shown. Expected sizes are as follows: purified ExoU (from P. aeruginosa), 74 kDa; N2, 65 kDa; I1, 76 kDa; I2, 73 kDa; C3, 35 kDa; C2, 72 kDa; C1, 76 kDa. The faint band present in all lanes is a yeast antigen recognized nonspecifically by the ExoU antiserum.