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

Fig 6. Ply-NH attenuates virulence of SPN and enables host tolerance and an intracellular lifestyle in the lower respiratory tract.

Fig 6

(A) Survival time (h) of CD1 mice infected intra-nasally with 1.5×106 CFU of D39:Ply-H, D39:Ply-NH, D39:ΔPly (Ply deletion mutant), D39:PlyW433F (mutant strain expressing a non-hemolytic Ply) or serotype 1 ST306. n = 10 mice per group. p-values are from Log-rank test vs D39:Ply-H, with Bonferroni correction applied for multiple comparisons. (B) CFU in lungs immediately following infection (0 h, n = 5 mice per group) and in surviving mice from (A) at 168 h.p.i. p-values are from two-way ANOVA analysis with Dunnett’s multiple comparisons test vs D39:Ply-NH. (C) Histopathology of H&E stained lung tissue samples of mice infected with D39:Ply-H, D39:Ply-NH, or ST306 SPN strains at 24 h.p.i. Magnification: 20X. (D) Survival time (h) of CBA/Ca mice infected intra-nasally with 1×106 CFU of D39:Ply-H, D39:Ply-NH, D39:ΔPly, D39:PlyW433 or ST306. n = 10 mice per group. p-values are from Log-rank test vs D39:Ply-H, with Bonferroni correction applied for multiple comparisons. (E) Lung CFU at 96 h.p.i in surviving mice from the experiment performed in (D). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (F) (i) SBF-SEM and (ii) TEM images of mice lung sections following infection with ST306 for 24 h showing its intracellular localization (marked with arrow). Scale bar: 5 μm for (i) and 1 μm for (ii). (G) SPN CFU in bronchoalveolar lavage or post-lavage lung homogenates (tissue) of CBA/Ca mice at 48 h.p.i. For lavage, trachea were exposed and a syringe was used to perform 5×1 ml PBS with 1 mM EDTA washes of the lung. Pooled lavage was serially diluted onto blood agar to determine planktonic and weakly adhered pneumococcal CFU. Lavaged lungs were removed, homogenized and plated onto blood agar to determine intracellular or strongly-adherent pneumococcal CFU. The lines depict SPN CFU counts in bronchoalevolar lavage and post lavage lung homogenates from the same animal. p-values are from two-way ANOVA analysis with Tukey’s multiple comparisons test. Experiments in (A-G) were performed a single time, with the stated numbers of animals per experimental group.