Fig 6

Fig 6

A,B. Bafilomycin treatment increases susceptibility to WT and ΔO-Ag S. sonnei. Survival curves of larvae treated with control DMSO vehicle (blue) or Bafilomycin (red) upon infection in the HBV with WT (A) or ΔO-Ag (B) S. sonnei. Experiments are cumulative of 3 biological replicates. Bacterial input: ~7000 CFU. Statistics: Log-rank (Mantel-Cox) test; ****p<0.0001. C. ATP injections protect against S. sonnei infection. Survival curves of larvae injected in the HBV with control water (blue) or ATP (red) 3 hours prior to infection of the same compartment with S. sonnei. Experiments are cumulative of 3 biological replicates. Bacterial input: ~7000 CFU. Statistics: Log-rank (Mantel-Cox) test; ****p<0.0001. D. Bafilomycin treatment and ATP injections counteract each other. Survival curves of larvae injected in the HBV with ATP 3 hours prior to infection of the same compartment with S. sonnei and treatment with control DMSO vehicle (blue) or Bafilomycin (red). Experiments are cumulative of 3 biological replicates. Bacterial input: ~7000 CFU. Statistics: Log-rank (Mantel-Cox) test; ****p<0.0001. E,F. S. sonnei O-Ag is required to counteract acidification-mediated clearance by human neutrophils.ΔO-Ag (grey), complemented strain (ΔO-Ag^+pSSO-Ag, blue) or WT (red) S. sonnei were incubated with peripheral human neutrophils and exposed to DMSO (vehicle control treatment, E) or Bafilomycin (F). Difference in bacterial killing was quantified by plating from lysates of infected neutrophils at 1 hpi. Experiments are cumulative of 3 biological replicates from 3 independent donors. Statistics: one-way ANOVA with Sidak’s correction; ns, non-significant; *p<0.0332; **p<0.0021. G,H. Model of S. sonnei O-antigen counteracting neutrophils in vivo. Upon phagocytosis of WT S. sonnei (green), zebrafish neutrophils (red) rapidly acidify phagolysosomes containing bacteria. However, S. sonnei can tolerate this environment because of its O-antigen. S. sonnei replication leads to neutrophil and host death (G). S. sonnei without O-antigen fails to counteract acidification of neutrophil phagolysosomes. In this case, neutrophils clear infection and the host survives (H).