Orientia tsutsugamushi Modulates RIPK3 Cellular Levels but Does Not Inhibit Necroptosis

Figures

• 

  Figure 1. Ank1 and Ank6 bind and lower RIPK3 levels in an F-box-independent manner. (A) Schematics of Ank1, Ank6, and vIRD. For Ank1 and Ank6, the number that follows the underscore indicates the specific paralog of the multicopy paralogous family used in this study. Hereafter, the paralog indicator will be excluded. The ankyrin repeats (ARs; blue arrows), intervening sequence region (ISR; orange box), PRANC (pox protein repeats of ankyrin-C-terminal) domain (gray box), and F-box (green box) are indicated, as are the amino acids that encompass each region. (B–F) HeLa cells were transfected to express HA-RIPK3 and Flag-BAP, the indicated Flag-Ank proteins, or mock-transfected. (B) Whole-cell lysates were analyzed by immunoblot with antibodies against HA, Flag, RIPK1, and GAPDH. The HA:GAPDH (C) and RIPK1:GAPDH (D) densitometric signal ratios per sample were calculated. Asterisks indicate statistical significance compared to Mock + HA-RIPK3 (black) or Flag-vIRD + HA-RIPK3 (red). (E) Whole-cell lysates (Input) and eluates following anti-Flag co-immunoprecipitation were analyzed by immunoblot with the indicated antibodies. (F) The HA:GAPDH densitometric signal ratio per sample on the input…

• 

  Figure 2. RIPK3 levels are decreased in O. tsutsugamushi-infected endothelial cells. EA.hy926 cells were mock-infected (U) or infected (I) with O. tsutsugamushi. (A) Whole-cell lysates harvested at 24, 48, and 72 hpi were analyzed by immunoblot with antibodies against RIPK3, GAPDH, and O. tsutsugamushi TSA56. (B) The RIPK3:GAPDH densitometric signal ratio per sample was calculated and normalized to the respective uninfected condition per timepoint. Data are presented as mean ± standard deviation of three independent experiments. Student’s t-test was used to test for significant differences between uninfected and infected conditions at each timepoint. Statistically significant values are indicated as *** p < 0.001.

• 

  Figure 3. Uninfected and infected EA.hy926 cells resist TBZ-induced necroptosis. EA.hy926 cells were mock-infected (U) or infected with O. tsutsugamushi (I). At 72 hpi, cultures were treated with vehicle (Veh) or TBZ (1 h pretreatment with 1 μM BV6 + 20 μM zVAD-fmk followed by 10 ng mL−1 TNF for 4 h). Cells were then incubated with PI and FITC-Annexin V and analyzed by flow cytometry. The mean percentage of cells per quadrant ± one standard deviation was calculated and depicted in representative dot plots (A). Quadrant 1 (Q1; FITC-/PI+) and Q2 (FITC+/PI+) cells are necroptotic and dead, respectively. Q3 (FITC+/PI-) cells are early apoptotic. Q4 (FITC-/PI-) cells are alive. The combined percentages of dead and necroptotic (Q1 and Q2) cells per sample are displayed in (B). Data in (B) are presented as mean ± standard deviation values of six experiments. One-way ANOVA with Tukey’s post hoc test was used to test for significant differences between all samples. Statistically significant values are indicated as * p < 0.05.

• 

  Figure 4. O. tsutsugamushi sensitizes HeLa cells to necroptosis induced by RIPK3 overexpression. (A,B) HeLa cells were transfected to express HA-RIPK3 for 16–24 h then mock-infected (U) or infected with O. tsutsugamushi (I). Whole-cell lysates harvested at 24, 48, and 72 hpi were analyzed by immunoblot (A) with antibodies against HA, phosphorylated (p)-RIPK3, MLKL, p-MLKL, TSA56, and GAPDH. (B) Densitometric signals of HA, p-RIPK3, MLKL, and p-MLKL normalized to GAPDH were calculated per sample and normalized to the corresponding uninfected sample at each timepoint. (C,D) U and I HeLa cells were transfected to express Flag-BAP or Flag-RIPK3 at 24 hpi. At 48 hpi, cells were incubated with PI and FITC-Annexin V and analyzed by flow cytometry. The mean percentage of cells per quadrant ± one standard deviation was calculated and depicted in representative dot plots (C). Q1 (FITC-/PI+) and Q2 (FITC+/PI+) cells are necroptotic and dead, respectively. Q3 (FITC+/PI-) cells are early apoptotic. Q4 (FITC-/PI-) cells are viable. The combined percentages of dead and necroptotic (Q1 and Q2) cells per sample are displayed in (D). Results are representative of three independent experiments. Data are…

• 

  Figure 5. Endogenous MLKL does not co-localize with O. tsutsugamushi or L. monocytogenes. O. tsutsugamushi-infected HeLa cells (A,B), L. monocytogenes-infected HT-29 cells (C,D), and uninfected controls were fixed in ice-cold methanol and immunolabeled with antibodies against MLKL (A,C), O. tsutsugamushi TSA56 (A), and L. monocytogenes (C) and examined by immunofluorescence microscopy. Representative immunofluorescence micrographs are presented in panels (A,C). (B,D) Relative fluorescence intensity plots of green and red pixels from immunofluorescence micrographs of O. tsutsugamushi (A) or L. monocytogenes-infected (C) cells normalized to the highest fluorescence intensity per channel moving left to right along the white lines. Scale bars = 10 μm (A) or 20 μm (C). Data are representative of three independent experiments.