Fig 1: BopE targets the Rab32-dependent defense pathway allowing B. pseudomallei to survive in the host. (A) Representative immunofluorescence images of co-localization of Rab32 and B. pseudomallei mutant strains in RAW264.7 cells at the indicated times after infection. Scale bar, 50 µm. (B) Quantification of B. pseudomallei in Rab32-positive vesicles in panel A. Ten random fields were counted in each slice, and three different experiments were conducted in every group. All assessments were performed in a blinded manner. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA). (C and D) Intracellular B. pseudomallei burdens in RAW264.7 cells at 8 hpi. WT is the wild-type strain of B. pseudomallei, ΔbopE is the bopE deficient strain, and ΔbopE/bopE is the bopE complementation strain. The data were showed as means ± SD. Three different experiments were conducted in each group. *P < 0.05 and **P < 0.01 (one-way ANOVA). (E) Survival rate of WT, ΔbopE, or ΔbopE/bopE B. pseudomallei-infected BALB/c mice. Mice models were infected with 4 × 105 CFU of B. pseudomallei WT, ΔbopE, or ΔbopE/ bopE and then continued to be observed until day 5. χ2 = 4.101, P = 0.0429 (ΔbopE vs. WT); χ2 = 5.141, P = 0.0234 (ΔbopE vs. ΔbopE/bopE), 12 mice per group (log-rank test). (F) The graphical model illustrates that BopE, a T3SS-3 effector of B. pseudomallei, suppresses the Rab32-dependent defense pathway in macrophage, thereby facilitating intracellular replication.
Fig 2: BopE interferes with the nucleotide exchange process of Rab32. (A) Nucleotide exchange assay for Rab32. ΔFluorescence is equal to fluorescence intensity measured at each specified time point minus the baseline fluorescence intensity recorded at the onset of the experiment. All dots are presented as mean ± SD and derived from three independent experiments. ***P < 0.001 (two-way ANOVA with Tukey’s method). (B) BopE inhibits the activation of Rab32 independent of the GEF activity. The mutant BopEN224P/R230Q exhibits high catalytic activity, while the mutant BopER207E/N216P has lost its catalytic activity. Fluorescence spectrometry assay data were showed mean ± SD from three independent experiments. ***P < 0.001 (two-way ANOVA with Tukey’s method). (C) BopE interacts with Rab32 independent of its GEF activity. Representative immunofluorescence images of co-localization of Rab32 and the GEF activity mutants of BopE in HEK293T cells. Rab32 was stained with an anti-Flag antibody (red), BopE, or its mutants tagged with GFP (green). Scale bar, 5 µm. (D) Confocal microscopy of BopE and Rab32 in panel C with fluorescence intensity plotted along the arrows.
Fig 3: B. pseudomallei T3SS effector protein BopE directly interacted with Rab32. (A) Co-immunoprecipitation assays of Rab32 and B. pseudomallei T3SS effector proteins. The molecular mass of FLAG-tagged T3SS effector proteins and that of GFP-tagged Rab32 are denoted within corresponding parentheses. “Input” refers to the total protein lysate, while “IP:Flag” indicates the immunoprecipitated protein using an anti-Flag antibody. “IB:GFP/Flag/Tubulin” denotes the immunoblotting with antibodies against GFP (to detect pEGFP-Rab32), Flag (to detect the T3SS effector proteins), and tubulin (as a loading control). (B) The interaction assay between B. pseudomallei T3SS effector proteins and Rab32 using yeast two-hybrid system. The transformants were plated onto low-stringency selection plates (left) to verify the successful integration of the plasmids and high-stringency selection plates (right) to detect any potential interactions between the T3SS effectors and Rab32. (C) Representative immunofluorescence images of co-localization of Rab32 and BopE in HEK293T cells. GFP-BopE and Flag-Rab32 were co-expressed in HEK293T cells. Rab32 was stained with anti-Flag (red), BopE, or SopD2 tagged with GFP (green). Images show maximum-intensity projections of confocal Z-stacks. The empty vector of pEGFP-C1 was used as a negative control, and SopD2 was used as a positive control. Scale bars, 5 µm. (D) Confocal microscopy of BopE and Rab32 in panel C with fluorescence intensity plotted along the arrows.
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