A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication (2021)
Figure 6

Figure 6. BspF promotes recruitment of TGN-derived membranes to rBCVs in an Arf6-/Rab8a-dependent manner.

(A) Representative confocal micrographs of BMMs infected with either wild type (2308), ∆bspF or or complemented ∆bspF (∆bspF::bspF) bacteria (red) for 24 h and immunostained for the TGN vesicular marker Stx6 (green) and GM130 (blue). Scale bars, 10 µm and 2 µm (insets). Magnified insets show the association between Stx6-positive vesicles and rBCVs.

(B) Recruitment of Stx6-positive vesicles to rBCVs (expressed as percentage of Stx6-positive BCVs) in BMMs infected for 24 h with either wild type (2308), ∆bspF or complemented ∆bspF (∆bspF::bspF) bacteria. Data are means ± SD from n=3 independent experiments, in which at least 300 BCVs were analyzed per experiment via CellProfiler image analysis. Asterisks indicate statistically significant differences compared to 2308-infected BMMs as determined by one-way ANOVA with Tukey's multiple comparisons test (P < 0.05).

(C) Representative Western blot analysis of Arf6, Rab8a, Rab6a/a' and Stx6 depletions in BMMs following siRNA-mediated knockdowns, compared to non-targeting siRNA (siNT) treatments. β-actin was used as loading control.

(D-F) Recruitment of Stx6-positive vesicles to rBCVs in BMMs treated with non-targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E) or Rab6a/a' (siRab6a/a') (F) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions; ns, not significant.

(G) Quantification of CTxB retrograde transport in BMMs following siRNA-mediated depletion of Stx6 (siStx6) after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. The asterisk indicates a statistically significant difference (P=0.0114, unpaired Student's t test) compared to the siNT control.

(H) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.

(I) Model of BspF remodeling of TGN-RE membrane traffic. Bacterially delivered BspF targets RE membranes where it binds ACAP1 and promotes inactivation of Arf6. Increased turnover of active Arf6 results in inhibition of the Arf6/Rab8a cascade and retrograde RE-TGN transport, which alters TGN-derived vesicular traffic and redirects Stx6-positive vesicles to rBCVs in a process that promotes intravacuolar bacterial growth.

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