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. 2020 Nov 20;16(11):e1009016. doi: 10.1371/journal.ppat.1009016

Fig 4. Abrogation of pore forming ability confers improved internalization capability in SPN strains harbouring Ply-NH.

Fig 4

(A-B) Invasion efficiency of R6:Ply-H and R6:Ply-NH strains in A549 cells (A) and THP-1 cells (B). (C-D) Invasion efficiency of R6:Ply-H, R6:Ply-NH (C) and D39:Ply-H, D39:Ply-NH and ST306 strains (D) in primary human pulmonary alveolar epithelial cells. (E) Comparison of invasion efficiency of R6:Ply-H and R6:Ply-NH following pre-treatment of A549 cells with purified recombinant Ply-NH and Ply-H (0.1 μg/ml), respectively. (F) Inhibition of internalization of R6:Ply-NH following pre-treatment of A549 cells with methyl β-cyclodextrin (Mβ-CD, 3 and 5 mM). (G) Dot blot showing localization of Ply-H and Ply-NH in low density lipid raft fractions of A549 cell membrane. CtxB and transferrin (Tfn) was used as positive and negative control, respectively. (H) Immunofluorescence image showing uptake of CtxB-FITC coated latex beads (1.1 μm) by A549 cells following pre-treatment with Ply-H and Ply-NH. Internalized beads are shown in red (arrow mark) while external beads are dual (yellow) colored. Scale bar: 5 μm. Data information: Experiments are performed thrice and data of representative experiments are presented as mean ± SD of triplicate wells. Statistical analysis was performed using Student’s two-tailed unpaired t-test (A-C; E-F) and one-way ANOVA with Tukey’s multiple comparison test (D). ns, non-significant; *p<0.05; **p<0.01; ***p<0.001.