Fig. 5: VgrG4 induces mitochondrial ROS production.

a Mitochondrial ROS (mtROS) was detected in infected A549 cells by confocal microscopy. Cells were treated with mitotracker green (50 μM, 30 min, in green), infected with YeVgrG4 for 90 min or with Kp52145 for 3 h, and treated with mitoSOX red mitochondrial superoxide indicator (10 µM for 30 min incubated in calcium free HBSS). Fluorescence was measured (b) in A549 cells upon infection with Y. enterocolitica strains for 90 min or K. pneumoniae strains for 3 h, followed by incubation with mitoSOX (10 µM incubated in calcium free HBSS for 30 min prior to measurement). c To demonstrate that VgrG4 localisation is important for ROS production, 2’,7’-dichlorofluorescein (DCF) was used to measure ROS production by A549 cells transfected with MFN2 siRNA or non-silencing (AS) control and infected with YeVgrG4 for 90 min. d DCF fluorescence was measured in A549 cells pre-treated with Mdivi-1 (10 µM, 2 h) or vehicle control (DMSO), and infected with YeVgrG4 for 90 min. Cells were treated with thapsigargin (1 μM), ruthenium red (100 μM), ryanodine (100 nM) 30 min before the end of infection. Mitochondria fragmentation was analysed by confocal microscopy of A549 cells treated with mitotracker red (50 μM, 30 min, in red) and stained with Hoechst (in blue). Cells were pre-treated with mitoTEMPO, a mitochondrial superoxide scavenger (10 μM, 2 h), and infected with Kp52145 for 3 h (e); at least 100 cells were analysed per sample. One-way ANOVA with Tukey’s multiple comparison test was applied for statistical significance (p values indicated in the graph).