Fig. 6: S. flexneri evades septin cage-based cell-autonomous immunity during infection in an OspC-dependent manner.

a, b Synergy between OspC and OspG. HeLa cells infected with GFP-expressing S. flexneri strains (MOI = 6, 2 h 40 min) were analyzed by immunofluorescence. a Representative confocal images (scale bar, 10 μm, n = 3 independent experiments). b The percentages of septin caged bacteria are shown as mean ± SEM from n = 1753 (WT), n = 833 (ΔospC), n = 799 (ΔospG), and n = 988 (ΔospC ΔospG) distributed in n = 3 independent experiments. c, d Complementation of S. flexneri ΔospC strains with catalytically active OspCs enables bacteria to escape from septin cages. Samples were prepared as described above. Representative confocal images (scale bar, 10 μm, n = 3 independent experiments) are shown (c). The percentages of septin caged bacteria are shown as mean ± SEM from n = 1494 (WT), n = 1398 (ΔospC), n = 2241 (ΔospC+pOspC1/C3-WT), and n = 1296 (ΔospC+pOspC1/C3-EH/AA) distributed in n = 3 independent experiments (d). e, f OspC and OspG synergistically promote intercellular spread of S. flexneri. Plaque formation assays were performed as described in the Methods section. Representative images (scale bar, 1 mm, n = 2 independent experiments) are shown. Quantification results of plaque area (n = 30 plaques per condition) are presented as mean ± SEM. g–i Intracellular growth of S. flexneri in HeLa cells. HeLa cells were infected with indicated S. flexneri strains. Bacterial load was determined at 0.5 (g), 2 (h), and 4 h (i) post-infection (normalized to 0.5 h). Data: mean ± SEM (n = 5 independent experiments). j A proposed model illustrating S. flexneri effectors OspCs and OspG synergistically counteract septin cage formation. OspCs destabilize SEPT9-containing octamers and block cage formation via direct ADP-riboxanation, while OspG inhibits cage assembly through septin ubiquitination in a mechanism dependent on CAND1 phosphorylation. Created in BioRender. Zhang, Y. (2025) [https://BioRender.com/bpts0kk]. Unless otherwise specified, statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (two-sided). P-values are denoted as: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.