Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle
Fig 5
Loss of pore forming ability facilitates prolonged intracellular persistence of SPN.
(A) Intracellular survival efficiency of SPN R6 strains expressing either Ply-H, Ply-NH or Ply-DM (H150Y+T172I) in A549s were calculated as percentage survival at indicated time points relative to 0 h. (B) Intracellular survival efficiency of SPN R6 strains expressing either Ply-H or Ply-NH in THP-1 macrophages. (C) Confocal micrographs showing association of SPN (blue) expressing Ply-H or Ply-NH with Gal8 or Ubq (red), LC3 (green) and LAMP1 (pink) in A549s at 10 h.p.i. DAPI (blue) has been used to stain A549 nucleus and SPN DNA. Arrows designate the bacteria shown in insets. Event was localized at z-stack number: 4 out of 7 (for R6:Ply-H_Gal8 image), 4 out of 7 (for R6:Ply-H_Ubq image), 4 out of 8 (for R6:Ply-NH_Gal8 image) and 7 out of 11 (for R6:Ply-NH_Ubq image). Scale bar: 5 μm. (D) Percent co-localization of Gal8 with SPN strains expressing either Ply-H or Ply-NH in A549s at indicated time points post-infection. (E) Percentage co-localization of Ubq with SPN strains expressing either Ply-H or Ply-NH in A549s at indicated time points post-infection. (F-G) Quantification of co-localization of LC3 with Gal8 (F) or Ubq (G) positive R6:Ply-H in A549s at 10 h.p.i. (H) Quantification of co-localization of SPN strains expressing either Ply-H or Ply-NH with Lysotracker in A549s at 18 h.p.i. Data information: Experiments are performed thrice and data of representative experiments are presented as mean ± SD of triplicate wells. n≥100 SPN per coverslip (D-H). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test (A) and Student’s two-tailed unpaired t-test (B, D-E, H). ns, non-significant; *p<0.05; **p<0.01; ***p<0.001.