Fig 1.
Expression of C. burnetii T4BSS-translocated effectors in mammalian HeLa cells.
(A) Immunofluorescence confocal images show the representative subcellular localization of the indicated GFP- or HA-tagged C. burnetii effector proteins (green) after ectopic expression in HeLa cells. For contrast, DAPI, Mitotracker, or anti-tubulin, anti-calnexin and anti-LAMP-1 antibodies, were used as marked. Scale bars represent 10mm. (B) Expression of the indicated GFP-tagged C. burnetii effectors was analyzed by immunoblot analysis. Protein extracts from transfected HeLa cells were separated by SDS-PAGE, transferred to PVDF membrane and probed with anti-GFP antibody and anti-tubulin as loading control. A representative immunoblot is shown out of three independent experiments with similar results.
Fig 2.
Expression of C. burnetii T4BSS-translocated effectors in yeast.
(A) Immunoblot with anti-GFP antibodies on yeast lysates from transformants of YPH499 strain with pYES2-GFP-derived plasmids expressing the indicated GFP-tagged C. burnetii effector proteins grown on SCRaf selective medium and induced for 4 h by addition of galactose. (B) Serial dilution drop assays to monitor growth under induction (Galactose) and control (Glucose) conditions of representative transformants as in (A). (C) Growth in liquid SCGal medium of yeast transformants expressing the indicated GFP fusions after 36 h of growth in 96-well plates. Data are the average of 36 wells per experiment, corresponding to three different transformant clones (12 wells per clone), and are expressed in percentage with respect to the control transformed with the empty vector (average O.D.600 of control samples was considered 100% growth). Bars correspond to the standard deviation. P-values noted were determined by the Student’s T-test.
Fig 3.
CaeA behaves like apoptotic inducers Ybh3 and human Bax in yeast.
(A) CaeA expression induces oxidative stress. The graph represents the enhancement of positive YPH499 yeast cells expressing the indicated C. burnetii effector proteins from the corresponding GAL1-driven expression vectors, as determined by flow cytometry, expressed as times-fold over control cells transformed with the empty vector. The fluorochromes propidium iodide (white bars), rhodamine 123 (grey bars) and dihydroethidium (black bars), respectively monitor cell death, altered mitochondrial potential and intracellular ROS accumulation in the yeast populations. GAL1-driven YBH3 overexpression on the same strain was used as a positive control. Data correspond to a representative experiment of triplicate replicas. (B) Flow cytometry histograms of representative experiments as in (A) on YPH499 transformants expressing the indicated proteins from the corresponding GAL1-driven expression vectors. The gating threshold is marked by a vertical line. The percentage of cells in the population beyond the gating threshold is shown for each histogram.
Fig 4.
GFP-CaeA primarily accumulates at JUNQs, and residually at IPODs in yeast cells.
(A) Fluorescence microscopy of GFP-CaeA-expressing yeast cells counterstained with DAPI and FM4-64 to visualize respectively nuclei and vacuoles. YPH499 cells transformed with pYES2-GFP-CaeA were induced in galactose for 6 h. Filled white arrowheads point to the nucleus-associated spot, normally the most intense; hollow arrowheads indicate the less intense vacuole-associated spot. (B) A prominent GFP-CaeA spot is associated to the nucleus. Confocal microscopy of representative cells displaying one only (left) or two (right) GFP-CaeA spots, as indicated. DAPI, which marks nuclear and mitochondrial DNA, is shown in red color for better contrast with green GFP-CaeA. The relative position of GFP-CaeA spots to nuclear DNA for each cell is depicted in the schemes at the bottom. (C) Representative micrograph of a pYES2-GFP-CaeA transformant of strain VHY87, bearing an integrated marker for the ER (HDEL-DsRed). The filled white arrowhead indicates the GFP-CaeA spot engulfed by the perinuclear ER, and the hollow arrowhead shows the spot located in the cytoplasm beyond the perinuclear ER. (D) Co-localization of GFP-CaeA (green) with the JUNQ marker Ubc9ts-DsRed (red). YPH499 yeast cells were co-transformed with pYES2-GFP-CaeA and pESC-LEU-CHFP-Ubq9ts and induced in galactose for 4 h. The GFP-CaeA positive cell in the upper part of the picture shows a representative cell with one single GFP-CaeA spot, which corresponds to the JUNQ (white filled arrowhead). In cells in which a second, less intense spot appears, it shows no co-staining with Ubc9Ts (hollow arrowhead, bottom cell). (E) Co-localization of GFP-CaeA (green) with the IPOD marker Rnq1 (red). YPH499 yeast cells were co-transformed with pYES2-GFP-CaeA and p425-GAL1-RNQ1-mCherry, and induced as in C. The second, less intense, GFP-CaeA spot is clearly associated to the Rnq1 marker. As in panels (C) and (D), filled white arrowheads point at GFP-CaeA at JUNQs, whereas empty arrows mark IPODs. Bars represent 5 μm in all panels.
Fig 5.
CaeA is ubiquitinated in yeast and mammalian cells.
(A) GST-CaeA was affinity purified from YPH499 cell lysates expressing the fusion protein from pEG(KG)-CaeA after 6h-galactose induction. A transformant with the empty plasmid expressing GST alone were analyzed in parallel as a control. Original inputs (Lysate) and the result of affinity purification on glutathione-coated beads (Pull-down) were solved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with both anti-GST (left) and anti-ubiquitin (right) antibodies, as indicated. (B) HEK293T cells were co-transfected with plasmids expressing the indicated GFP-fusion proteins and HA-ubiquitin. GFP-tagged proteins were immunoprecipitated (IP) with a rabbit anti-GFP antibody. Lysates and IPs were solved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with both anti-GFP (top) and anti-HA antibodies (bottom).
Fig 6.
Localization of GFP-AnkA to the yeast IPOD compartment.
(A) YPH499 transformants expressing GFP-AnkA (green) from the pYES2-GFP-AnkA plasmid after induction in galactose for 4 h were incubated with FM4-64 (red) for 30 min to allow visualization of the vacuolar membrane. (B) YPH499 yeast cells were co-transformed with pYES2-GFP-AnkA and p425-GAL1-RNQ1-mCherry, an induced as above. Representative cells are shown of two typical patterns: neat co-localization of both red and green channels (upper panel) and inclusion of the green GFP-AnkA signal within a larger Rnq1-mCherry compartment (lower panel). Bars represent 5 μm.
Fig 7.
Localization of CBU0077 and AnkB in yeast cells.
(A) GFP-CBU0077 is associated to yeast vacuolar membranes. Fluorescence microscopy of YPH499 transformants expressing GFP-CBU0077 (green) from the pYES2-based expression vector and induced with galactose for 4 h prior to incubation with FM4-64 (red) as in Fig 5A. Arrowheads mark vacuoles with membranes clearly marked in both channels. (B) GFP-CBU0077 accumulates at abnormal cytoplasmic structures that harbor the ER marker HDEL. Fluorescence microscopy of pYES2-GFP-CBU0077 transformants of strain VHY87, constitutively expressing the ER marker HDEL-DsRed. The filled arrowhead in the upper panel points to a cell lacking GFP signal, serving as a visual reference for a typical yeast ER shape (red channel) consisting of neat perinuclear and plasma-membrane associated membranes. This pattern is typically distorted in the adjacent cell, showing high GFP-CBU0077 signal. In the middle panel, a characteristic cell in which some of the cytoplasmic bar-shaped cytoplasmic structures containing GFP-CBU0077 (green) co-localize with the ER marker (hollow arrowhead). In the lower panel, the arrowhead indicates a characteristic cell with a high expression level of GFP-CBU0077 strongly associated with the perinuclear ER. (C) Localization of AnkB to the yeast nucleus and association with the nucleolar compartment. YPH499 cells were co-transformed with pYES2-GFP-AnkB and pUN100-mCherry-NOP1, induced in galactose for 4h, co-stained with DAPI to counterstain nuclei and observed by fluorescence microscopy. Representative fields are shown displaying cells at different stages of the mitotic cycle. The GFP-AnkB signal (green) is enriched within the yeast nucleus at a spot either overlapping or adjacent to the mCherry-Nop1 nucleolar marker. Bars correspond to 5 μm in all panels.
Fig 8.
AnkB expression enhances S. cerevisiae sensitivity to aminoglycosides.
(A) Graph displaying the measurements of the growth inhibition halo (mm) around a 6 mm-disc (horizontal line) soaked with a solution of the hygromycin B or neomycin sulfate concentrations indicated on YPH499 cells bearing pYES2-GFP-AnkB or the empty vector as a control. Data are the average of five (hygromycin B) or two (neomycin) independent experiments performed on different transformants. (B) Dose-response curve showing the effect of ½ serial dilutions of neomycin on growth of YPH499 transformants bearing pYES2-GFP-AnkB or the empty vector, as a control, cultured in SCGal liquid medium. Data are the average or three biological replicates, each performed on an individual transformant clone. Asterisks denote statistical significance of data (*) p<0.05; (**) p<0.01; (***) p<0.005. P-values were calculated by the Student’s T-test.