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Fig. S1. Intracellular trafficking and O-antigen expression of Schu S4 mutants.

A and B. Confocal micrographs of BMMs infected with Francisella strains and stained for either LAMP1 (A) or cellular markers of autophagy (B) at 2 h post-infection. Wild-type Schu S4 (Schu S4), Schu S4ΔFTT1236 (ΔFTT1236) Schu S4ΔmanC (ΔmanC), Schu S4ΔfevR (ΔfevR) and Schu S4ΔdipA (ΔdipA) DNA was stained with DAPI (grey) and specific cellular markers are shown in red. Insets are indicated with a white box (A) or arrows (B) and magnified to show bacteria (Ft), and LAMP1, Ubiquitin (Ub), p62/SQSTM1 (p62) or LC3 in red. Scale bars, 10 and 2 μm.

C and D. Western blots of whole-cell lysates of wild-type Schu S4 (Schu S4) Schu S4ΔfevRfevR), Schu S4ΔdipAdipA). A total of 1 × 107 bacterial equivalents were resolved and probed for either LPS O-antigen (C) or O-antigen capsule (D), and GroEL was used as a loading control.

Fig. S2. Representative transmission electron micrographs of wild-type Schu S4 (Schu S4), Schu S4ΔFTT1236 (ΔFTT1236), Schu S4ΔmanCmanC) and Schu S4ΔfevRfevR), mutants at 1 h post-infection. Scale bar, 500 nm.

Fig. S3. Capture of Francisella O-antigen mutants by autophagy in human macrophages.

A, C and E. Confocal micrographs of MDMs infected with wild-type Schu S4 (Schu S4), Schu S4 ΔFTT1236 (ΔFTT1236) or Schu S4ΔmanCmanC), showing recruitment of (A) Ubiquitin (Ub), (C) p62/SQSTM1 (p62) or (E) LC3 at 2 h post-infection around the O-antigen mutants stained with DAPI (grey), and specific cellular markers (red). Insets are indicated with arrows and magnified to show bacteria. Scale bars,10 and 2 μm.

B, D and F. Quantification of autophagy markers recruitment to Schu S4 (black), Schu S4ΔFTT1236 (red) and Schu S4ΔmanC (blue) with (B) Ubiquitin, (D) p62/SQSTM1 or (F) LC3. Data are means ± SD (n = 3).

Fig. S4. Phagosomal escape is required for autophagic targeting.

A. Colocalization of ubiquitinated wild-type Schu S4 (Schu S4), Schu S4ΔFTT1236 (ΔFTT1236) and Schu S4ΔmanCmanC) with the phagosomal marker LAMP1 at 2 h post-infection. Data are means ± SD (n = 3; asterisks indicate a P value < 0.05 by the unpaired Student's two-tailed t-test). ND, not detected.

B–E. Effect of bafilomycin-A1 (BAF) treatment on intracellular trafficking of O-antigen mutants. Recruitment of LAMP1 (B), Ubiquitin (C), p62/SQSTM1 (D) or LC3 (E) at 2 h post-infection to wild-type Schu S4, Schu S4ΔFTT1236 and Schu S4ΔmanC in BMMs treated with either no (white bars) or 100 nM (black bars) BAF. Data are means ± SD (n = 3; asterisks indicate a P value < 0.05 by the unpaired Student's two-tailed t-test).

Fig. S5. Autophagy induction of infected macrophages.

A. Quantification of LC3 puncta in uninfected BMMs (Mock) or BMMs infected with wild-type Schu S4 (Schu S4), Schu S4ΔFTT1236 (ΔFTT1236) and Schu S4ΔmanCmanC) at 2 h post-infection. Data are means ± SD (n = 3).

B. Quantification of LC3 puncta in uninfected BMMs subjected (black bar) or not (white bar) to amino acid starvation. Data are means ± SD (n = 3; asterisks indicate a P value < 0.01 by the unpaired Student's two-tailed t-test). Quantification of endogenous LC3 with wild-type Schu S4 (Schu S4), Schu S4ΔFTT1236 (ΔFTT1236) and Schu S4ΔmanCmanC) at 2 h post-infection in untreated (white bars) or starved (black bars) macrophages. Data are means ± SD (n = 3).

Table S1. Primers used in this study.

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