pubmed.ncbi.nlm.nih.gov

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1 - PubMed

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Ka-Wing Wong et al. PLoS Pathog. 2005 Oct.

Abstract

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.

PubMed Disclaimer

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activation of Rac1 in Single Cells as Demonstrated by FRET

(A) Schematic representation of intermolecular FRET from Rac1-GTP to PBD. In the absence of GTP loading, mCFP-Rac1 is unable to bind mYFP-PBD. Upon GTP loading (activation) of Rac1, mCFP-Rac1-GTP is able to bind to the downstream effector construction mYFP-PBD. This allows the emission from mCFP to excite mYFP, resulting in emission at 527 nm. (B) Display of fluorescence emission and sensitized FRET in individual transfectants. COS1 cells cotransfected with various mCFP-Rac1 derivatives and either mYFP-PBD or mYFP-PBD(LL) were subjected to FRET analysis, and sensitized FRET was calculated (Materials and Methods). Displayed are the channels for CFP and YFP emission as well as the relative Rac1 activation throughout the cell as determined by sensitized FRET. The color-scale bar represents the amount of Rac1 activation displayed as relative pixel intensity in noted area of cell (Materials and Methods). White bar represents 10 μm. (C) FRET is dependent on coexpression of active mCFP-Rac1 and mYFP-PBD. FRET from ten ROIs, which represented two cytoplasmic regions from each of five cells, were plotted as a function of the intensity of the mCFP-Rac1 donor. (D) Activation of Rac1 increases normalized levels of FRET. Data from (C) were normalized to amount of mCFP-Rac1 in each region of interest and displayed. Displayed are means ± standard errors of the mean (SEMs) (E) Enhanced emission of the mCFP-Rac1 donor resulting from acceptor photobleaching requires Rac1 activation. The acceptor mYFP-PBD was selectively photobleached for 2 min, and amount of mCFP-Rac1 donor emission was compared before and after irradiation (Materials and Methods). The percentage increase in emission of after irradiation is displayed. To determine emission enhancement, ten ROIs were analyzed. Displayed are means ± SEMs.

Figure 2
Figure 2. Localized Activation of Rac1 on the Phagosomal Membrane during Y. pseudotuberculosis Uptake

(A–D) Transfected COS1 cells were incubated with Y. pseudotuberculosis (YPIII[P−]) for 20 min, fixed, and then immunostained with anti-Y. pseudotuberculosis to detect partially internalized bacteria (Extracellular bacteria; see Materials and Methods). Rac1 activation was measured by sensitized FRET, and normalization (Normalized FRET) was performed by determining the amount of FRET at each pixel relative to the mCFP-Rac1 intensity at that pixel (Materials and Methods). Color-gradient scales, as described in Figure 1, were used to represent the intensity of signal for Rac1 activation (Sensitized FRET) as well as for normalized Rac1 activation (Normalized FRET). Note the scales are not identical in each window. Arrows indicate nascent phagosomes, as described (Materials and Methods), and these are simultaneously displayed in insets. Shown are cotransfection of: mCFP-Rac1(WT) and mYFP-PBD (A), constitutively active mCFP-Rac1V12 with mYFP-PBD (B), dominant negative mCFP-Rac1N17 with mYFP-PBD (C), and effector binding-defective mCFP-Rac1C40 with mYFP-PBD (D). White bar represents 10 μm. (E) FRET in response to Y. pseudotuberculosis adhesion is dependent on coexpression of active mCFP-Rac1 and mYFP-PBD. Sensitized FRET (Rac1 activation) at regions surrounding nascent phagosomes was plotted as a function of the intensity of the mCFP-Rac1 donor in the cell. (F) The pool of Rac1 surrounding incoming bacteria is preferentially activated. The intensity of FRET that surrounded nascent phagosomes was compared to that found in nearby cytoplasmic areas, normalized against the concentration of mCFP-Rac1 found in each area, and then used to calculate the ratio of normalized Rac1 activity of nascent phagosome to that of background (Materials and Methods). Data from 20 nascent phagosomes in cells expressing mYFP-PBD along with either mCFP-Rac1(WT) or mCFP-Rac1V12 were displayed as mean ± SEM (p = 0.006).

Figure 3
Figure 3. YopE Selectively Inactivates Membrane-Associated Rac1.

COS1 cells cotransfected with plasmids expressing mYFP-PBD and noted plasmids were challenged at 37 °C for 30 min with Y. pseudotuberculosis YP17(yopH −, yopT −, yopE −) (noted as “Control” in [A] and [B]) or YP17/pYopE (noted as “+YopE” in [A–C]). Bacteria were grown at 37 °C for 1 h to induce expression of the type III secretion system prior to infection (Materials and Methods). The C189S mutation blocks plasma membrane localization of Rac1, whereas R66A prevents extraction of Rac1 by RhoGDI from plasma membrane into cytosol. (A and B) Displayed are images of typical cells showing phase contrast, mCFP fluorescence and color-scaled sensitized FRET or normalized (Rac1 activation) for YP17 (A) or Yp17/pYopE (A and B) infected cells (Materials and Methods). White bar represents 10 μm. (C) Cytoplasmic Rac1 is not a YopE target. Normalized FRET was determined (Materials and Methods) by imaging cytoplasm of eight infected cells (Materials and Methods). Data for mCFP-Rac1 activation was normalized against mCFP-Rac1 intensity (normalized Rac1 activation) and displayed as mean ± SEM. *****, p < 5 × 10−6 between control cells and YopE-treated cells or between Rac1(WT) and Rac1C189S-transfected cells. NS, no significant difference between Rac1(WT)-transfected cells and Rac1R66A-transfected cells.

Figure 4
Figure 4. Y. pseudotuberculosis YopT Removes Multiple Rho Family Members from Nascent Phagosomes

(A–D) COS1 cells transfected with plasmids encoding T7-Rac1, T7-Rac1C189S, myc-Cdc42, myc-RhoA, or HA-Arf6 were incubated at 37 °C for 30 min with Y. pseudotuberculosis YP17 (A and C) or YP17/pYopT (B and D) grown under described conditions (Materials and Methods). (A and B) To identify nascent phagosomes, bacteria were probed as described (Materials and Methods), and small GTPases were identified by immunoprobing against appropriate tags. Pink identifies the extracellular region of a bacterium and blue represents the internalized region. Green indicates the staining for small GTPases. (C and D) Quantifications of the percentage (mean ± SEM in triplicate) of partially internalized bacteria that stained positively for Rac1 (n = 33), Cdc42 (n = 33), RhoA (n = 33), Rac1C189S (n = 33), or Arf6-HA (n = 11). (E) Representative localization of uninfected cells expressing mYFP-Rac1(WT), mYFP-Rac1C189S, or mYFP-Rac16Q, as well as a mYFP-Rac1(WT) transfectant challenged with YP17/pYopT for 30 min (WT; +YopT). White bar represents 1 μm. (F) The presence of excess RhoGDI interferes with the activity of YopT. Y. pseudotuberculosis YP17/pYopT (+pYopT) was introduced for 30 min onto COS1 cells or transfectants overexpressing RhoGDI. Nuclear localization of Rac1 (defined as the ratio of the mean nuclear Rac1 intensity relative to the mean cytoplasmic Rac1 intensity) was determined in each case.

Figure 5
Figure 5. YopT Cleavage Does Not Prevent Rac1 Activation

COS1 cells were cotransfected with plasmids expressing mYFP-PBD and either mCFP-Rac1 or mCFP-Rac1C189S followed by exposure to Y. pseudotuberculosis for 30 min (Materials and Methods) and FRET analysis in cytoplasmic ROIs. (A–C) Cells expressing mCFP-Rac1(WT) challenged with either YP17 (control) (A) or mCFP-Rac1(WT) challenged with YP17/pYopT (+YopT) (B). Rac1 activation is maintained in the presence of YopT cleavage (C). Data for normalized Rac1 activity from eight cells described in (A) and (B) were quantitated as mean ± SEM. (D) Cytoplasmic localization of Rac1 does not affect activation. FRET was analyzed in ROIs from cytoplasmic regions of uninfected cells transfected with mCFP-Rac1(WT) or mCFP-Rac1C189S and normalized versus amount of mCFP observed in each cell. NS, no significant difference between control and experimental. (E) Example of normalized FRET observed in uninfected cell expressing mCFP-Rac1C189S. White bar represents 10 μm.

Figure 6
Figure 6. Overexpression of YopT Interferes with YopE-Mediated Inactivation of Rac1 by Blocking YopE Translocation

COS1 cells were cotransfected with plasmids expressing mYFP-PBD and mCFP-Rac1, challenged for 30 min with Y. pseudotuberculosis strains YP15 (yopE +), YP15/pYopT (yopE +yopT +) or YP17/pYopT (yopE −yopT +) and processed for FRET analysis as in Figure 5 (Materials and Methods). (A) Typical FRET images of cells incubated with denoted strains. (B) The presence of pYopT interferes with the ability of YopE to inactivate cytoplasmically localized Rac1. Data represent normalized FRET, using ROIs within the cytoplasms of ten cells. ***** p < 5 × 10−5; * p = 0.01. Normalized FRET was determined as described (Materials and Methods). (C) Plasmid-expressed YopT promotes translocation of active Rac1 into the nucleus in the presence of YopE. Nuclear accumulation of Rac1 was determined by presence of mCFP-Rac1 fluorescence (bar graph), and activation was determined in that population showing accumulation using FRET analysis of nuclear regions (data below bar graph; Materials and Methods). (D) Overexpression of YopT blocks translocation of YopE from bacteria into host cells. After 1 h of infection with Y. pseudotuberculosis strains at MOI = 50, 0.5 × 106 COS1 cells were extracted with 0.1% NP-40 and separated into soluble and pellet fractions to identify translocated YopE in the soluble fraction (Materials and Methods). One-sixth of the soluble fraction and one-half of the pellet were analyzed by immunoblotting using an anti-YopE antibody. Strains used were as in (A), with the addition of the yscU mutant, defective for type III secretion as a negative control.

Figure 7
Figure 7. Temporally Regulated Interference of YopE Activity by YopT

COS1 cells cotransfected with mCFP-Rac1 and mYFP-PBD were incubated with the noted strains and processed for FRET analysis using ROIs located within the cytoplasm as in Figure 6 (Materials and Methods). (A) YopE can inactivate cytosolic Rac1 in the presence of YopT. FRET was determined after 30 min of infection with COS1 cells for the following strains. E, YP15 (yopT −yopE +); IP32953, clinical isolate expressing both YopT and YopE; T, YP17/pYopT2 (yopT +yopE −); and E + T, mixed infection of YP17/pYopT2 (yopT +yopE −) and YP15 (yopT −yopE +) added simultaneously to cells. (B) Injection of YopT prior to YopE interferes with Rac1 inactivation by YopE. Control → E, cells were infected with YP17 for 30 min and then incubated with YP15 (yopE +) for 30 min; IP32953, cells were incubated with clinical strain IP32953 (yopT +yopE +) for 1 h; and T → E, YP17/pYopT2 (yopE −) was introduced onto cells for 30 min prior to incubation of YP15 (yopE +) with cells for 30 min more. ***** p < 5 × 10−5; ** p = 0.002. Results from eight cells were quantitated as mean ± SEM. (C) Examples of sensitized FRET and normalized FRET for cells infected with denoted strains, as above. (D) YopT-promoted Rac1 nuclear localization occurs after YopE pretreatment. COS1 cells were incubated with bacteria using the above conditions, with the added protocol of incubating the cells with bacteria expressing YopE for 30 min prior to infection with bacteria that express YopT. E → T, YP15 (yopE + yopT −) was introduced onto cells for 30 min, prior to incubation of YP17/pYopT2 (yopT + yopE −) with cells for 30 min more. Results are displayed from the analysis of ROIs in eight to ten cells, with measurement of the nuclear localization index for Rac1 (ratio of Rac1 in the nucleus to that in the cytoplasm; Materials and Methods). (E) Translocation of YopE is unaffected by the presence of YopT in either a serogroup I strain or using coinfection conditions. Infection conditions were as indicated above; translocation was assayed, and the amount of protein was determined by immunoblotting using an anti-YopE antibody in an NP-40 extractible fraction (Materials and Methods). Pellet, protein not extracted by NP40 (associated with bacterial pellet); Soluble, NP40-soluble fraction (translocated).

Figure 8
Figure 8. Nuclear Localization of Rac1 Protects It from Inactivation by YopE

COS1 cells expressing the probes for Rac1 activity (mCFP-Rac1 and mYFP-PBD) were challenged with the indicated strains and then analyzed for nuclear-localized Rac1 activation using FRET. To account for differences in PBD and Rac1 concentrations in the nucleus relative to the cytoplasm, FRET was normalized to both proteins (Materials and Methods). (A) The FRET readout in the nucleus requires Rac1 activation. Translocation of various mutants of mCFP-Rac1 was promoted by incubating COS1 cells with YP17/pYopT2 for 30 min. (B) YopE is not able to reverse or prevent the accumulation of active nuclear Rac1 promoted by YopT. Control, 30-min incubation with YP17 (yopE −yopT −); Control → E, 30-min incubation with YP17 (yopE −yopT −) followed by 30-min incubation withYP15 (yopE +yopT −); E + T, 30-min coinfection withYP15 (yopE +yopT −) and YP17/pYopT2 (yopT +yopE −); T, 30-min incubation with YP17/pYopT2 (yopT +yopE −); and T → E, 30-min incubation with YP17/pYopT2 (yopE −) followed by 30-min incubation withYP15 (yopE +yopT −). ***** p < 5 × 10−5 relative to control incubation. (C) IP32953 inactivates cytosolic Rac1 and activates nuclear Rac1. Two examples of cells infected with IP32953 for 1 h show intense Rac1 activity (displayed as Nuclear FRET) that corresponds to the nucleus shown in phase contrast images.

Similar articles

Cited by

References

    1. Isberg RR, Barnes P. Subversion of integrins by enteropathogenic Yersinia . J Cell Sci. 2001;114:21–28. - PubMed
    1. Pepe JC, Miller VL. Yersinia enterocolitica invasin: A primary role in the initiation of infection. Proc Natl Acad Sci U S A. 1993;90:6473–6477. - PMC - PubMed
    1. Marra A, Isberg RR. Invasin-dependent and invasin-independent pathways for translocation of Yersinia pseudotuberculosis across the Peyer's patch intestinal epithelium. Infect Immun. 1997;65:3412–3421. - PMC - PubMed
    1. Isberg RR, Leong JM. Multiple β1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell. 1990;60:861–871. - PubMed
    1. Clark MA, Hirst BH, Jepson MA. M-cell surface β1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells. Infect Immun. 1998;66:1237–1243. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources