Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eucaryotic cells

Abstract

YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of Yersinia binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known. Yersinia pestis YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of Y. pestis YopM in a Saccharomyces cerevisiae model previously found to support nuclear localization of YopM from an enteropathogenic Yersinia strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001). Y. pestis YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in S. cerevisiae that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among Yersinia YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by Yersinia infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because Y. pestis carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.

FIG. 1.

Structure of YopM and cartoon of yEGFP-YopM. (Top) Ribbon model of YopM based on PDB structure 1JL5 (12) modified by adding free-form lines to indicate residues at the beginning of the leader domain (red, at left) and at the end of the tail domain (red, at right) which were not resolved in the crystal structure. Coloring has been added to provide a visual aid for locating regions deleted in the various yEGFP-YopM molecules tested in this study (Table 1). (Bottom) Cartoon of yEGFP-YopM with numbered LRRs and the same coloring as in the ribbon model. The figure was generated using Swiss PBD Viewer and rendered with PovRay before final construction in Microsoft Powerpoint. It was printed from Adobe PhotoShop 6.0.

FIG. 2.

YopM localizes to the nucleus in yeast. S. cerevisiae RCD224 was induced for expression of yEGFP-YopM or yEGFP alone by growth in the presence of galactose. (A) The yeast DNA was stained with Hoechst 33342, and blue Hoechst fluorescence is shown in the left panel for the same cells pictured at right, where green yEGFP-YopM fluorescence is shown. Arrows point to nuclei. Note that some cells visible by Hoechst staining (lower left quadrant) are not expressing yEGFP-YopM strongly enough for detection. (B) The localization of green fluorescence is compared for S. cerevisiae RCD224 expressing yEGFP-YopM and yEGFP alone from the vector used to carry the yEGFP-YopM fusion proteins in this study. This and other figures were rendered by using Adobe Photoshop 6.0.

FIG. 3.

Efficient localization of YopM to the yeast nucleus requires functional vesicular trafficking. (A) yEGFP-YopM was expressed in S. cerevisiae RCD224 (wild type) and a temperature-sensitive sec18-1 S. cerevisiae strain. The localization of green fluorescence is compared for the two strains at the permissive temperature of 26°C, where both strains showed nuclear localization of YopM, and at the nonpermissive temperature for the sec18-1 strain (37°C), where nuclear localization occurred only in the wild-type yeast. (B) sec18-1 S. cerevisiae expressing yEGFP-YopM for 2 h was shifted to the nonpermissive temperature of 37°C for 5 h and then kept at 37°C for a further 2 h (left panel) or shifted back to the permissive temperature for 2 h (right panel) to determine if the inhibition of nuclear localization was reversible.

FIG. 4.

Two YopM proteins deficient for pathogenic function still localize in the yeast nucleus. (A) Immunoblot analysis of proteins extracted from S. cerevisiae RCD224 after induction by galactose. Lanes: kDa, molecular mass markers; —, vector control expressing only yEGFP; YM, yEGFP-YopM; 4-7, yEGFP-YopM ΔLRR4-7; 7-10, yEGFP-YopM ΔLRR7-10. The blots were probed with a rabbit polyclonal antibody against YopM; hence, yEGFP alone and background yeast proteins are not visualized. The top bands in the other lanes represent the full-length yEGFP-YopM proteins. (B) The distribution of green fluorescence is shown for yEGFP-YopM ΔLRR4-7 (Δ 4-7) and yEGFP-YopM ΔLRR7-10 (Δ 7-10), expressed in S. cerevisiae RCD224.

FIG. 5.

YopM ΔLRR4-7 and YopM ΔLRR7-10 also localize to the nucleus in HeLa cells after delivery by Y. pestis infection. HeLa cells were infected for 4 h with YopM⁻ Y. pestis KIM8-3233 or Y. pestis KIM8-3233 expressing YopM, YopM ΔLRR1-2, YopM ΔLRR1-4, YopM ΔLRR4-7, or YopM ΔLRR7-10. After a brief trypsin treatment, the infected cells were lysed with water to obtain the soluble cellular fraction (cytosol). YopM proteins that had been delivered to the HeLa cytosol by the surface-adherent yersiniae were visualized by immunoblot analysis and probed with a rabbit polyclonal antibody raised against the whole YopM, which recognizes all of the YopM proteins being tested. Lanes: YopM, wild-type YopM; Δ1-2, YopM ΔLRR1-2; Δ1-4, YopM ΔLRR1-4; Δ4-7, YopM ΔLRR4-7; Δ7-10, YopM ΔLRR7-10. (B) The distribution of YopM protein in the infected HeLa cells was determined by indirect immunofluorescence. The primary antibody was the rabbit polyclonal used in panel A; the secondary antibody was conjugated to the Oregon Green fluorochrome. The upper panels show Oregon Green fluorescence from an optical slice obtained by laser scanning confocal microscopy; the lower panels show the differential interference contrast image of the same cells. From left to right, the panels illustrate two, two, five, and six infected HeLa cells. The arrows on some of the cells point to the kidney bean-shaped nucleus that lies to one side of each cell.

FIG. 6.

Both halves of YopM localize to the nucleus in yeast. (A) A control test showing the localization of yEGFP-YopM and yEGFP alone compared for S. cerevisiae RDC224 (wild type) (top) and the protease-deficient S. cerevisiae y604pep4 (bottom). (B) Immunoblot analysis of proteins extracted from S. cerevisiae y604pep4 after induction by galactose. Lanes: —, vector control expressing only yEGFP; YM, yEGFP-YopM; N, N-terminal half of YopM fused to yEGFP (yEGFP-YopM ΔLRR8-end); C, C-terminal half of YopM fused to yEGFP (yEGFP-YopM Δaa19-LRR7). The blots were probed with a rabbit polyclonal antibody against YopM; hence, yEGFP alone and background yeast proteins are not visualized. (C) The distribution was determined for fluorescence from yEGFP-YopM ΔLRR8-end (the N-terminal half of YopM) and yEGFP-YopM Δaa19-LRR7 (the C-terminal half of YopM) in S. cerevisiae y604pep4. From left to right, yEGFP indicates fluorescence from yEGFP-YopM proteins, DAPI indicates fluorescence from nuclei (DNA) stained with DAPI, CPY indicates fluorescence from CPY in the vacuole, and Merge indicates overlay of the green, blue, and red channels. In the Merge panel, note that the green fluorescence of yEGFP now appears turquoise due to overlap with blue DAPI fluorescence in the nucleus and that there is no overlap of yEGFP-YopM-related fluorescence with the red CPY fluorescence in the vacuole.

FIG. 6.

Both halves of YopM localize to the nucleus in yeast. (A) A control test showing the localization of yEGFP-YopM and yEGFP alone compared for S. cerevisiae RDC224 (wild type) (top) and the protease-deficient S. cerevisiae y604pep4 (bottom). (B) Immunoblot analysis of proteins extracted from S. cerevisiae y604pep4 after induction by galactose. Lanes: —, vector control expressing only yEGFP; YM, yEGFP-YopM; N, N-terminal half of YopM fused to yEGFP (yEGFP-YopM ΔLRR8-end); C, C-terminal half of YopM fused to yEGFP (yEGFP-YopM Δaa19-LRR7). The blots were probed with a rabbit polyclonal antibody against YopM; hence, yEGFP alone and background yeast proteins are not visualized. (C) The distribution was determined for fluorescence from yEGFP-YopM ΔLRR8-end (the N-terminal half of YopM) and yEGFP-YopM Δaa19-LRR7 (the C-terminal half of YopM) in S. cerevisiae y604pep4. From left to right, yEGFP indicates fluorescence from yEGFP-YopM proteins, DAPI indicates fluorescence from nuclei (DNA) stained with DAPI, CPY indicates fluorescence from CPY in the vacuole, and Merge indicates overlay of the green, blue, and red channels. In the Merge panel, note that the green fluorescence of yEGFP now appears turquoise due to overlap with blue DAPI fluorescence in the nucleus and that there is no overlap of yEGFP-YopM-related fluorescence with the red CPY fluorescence in the vacuole.

FIG. 7.

The leader and tail of YopM tested alone do not concentrate preferentially in the yeast nucleus. (Left) The ability of the 32-residue tail of YopM to conduct yEGFP into the nucleus was tested by determining the fluorescence distribution for yEGFP-YopMTail in S. cerevisiae RCD224. (Right) Fluorescence distribution from yEGFP-YopMleader-HA in S. cerevisiae y604pep4.