Figure 6 | Scientific Reports

Figure 6

From: Invasion of Human Retinal Pigment Epithelial Cells by Porphyromonas gingivalis leading to Vacuolar/Cytosolic localization and Autophagy dysfunction In-Vitro

Figure 6The alternative text for this image may have been generated using AI.

High internalization and intracellular content of Pg strains within ARPE cells. (A–G) Transmission electron microscopy (TEM) of human ARPE cells infected with all fimbriated Pg strains (10 MOI) for 24 hours. P. gingivalis and mutant strains internalized retinal pigment epithelial cells. The sections show the intra-and extra-cellular contents of un-infected control (A), Pg381 (B,C), MFI (DE) and DPG3 (F,G) infected ARPE cells for 24 hours with 10 MOI (2 sets of representative images from each group). Orange arrows indicate several epithelial junctions such as long and short tight junctions between ARPEs and loss of integrity (B). Yellow arrows indicate Pg strains freely occupy the cytoplasm (B,C,E–G). The top and bottom sections show the different magnifications of randomly selected sections (Scale bar:- A-B: 2 µm, C-E, G: 1 µm, F, H: 0.5 µm). Refer the low magnification in Fig. S5 for the complete view. (H,I) Antibiotic Protection Assay: The figure shows the survival of both adhered and internalized (H), and only internalized (I) P. gingivalis strains after 24 hours of infection. ARPE cells infected with Pg381 or isogenic mutant strains and incubated with or without antibiotics were lysed and the survived extra (cell surface/membrane attached) and intracellular bacteria were re-suspended and maintained in anaerobic broth for 3 days. (H) The data represents CFU within and on the surface of ARPEs harvested from biological triplicates before antibiotic treatment. The experiments were repeated thrice with three technical replicates (n = 9). The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within three different experiments (n = 3) with 3 technical replicates (n = 3) from each experiments. (n = 9; ****P < 0.0001). CFU - Colony Forming Unit. (I) Invasion efficiency of Pg strains were expressed as the percentage of the initial inoculum recovered after antibiotic treatment. The means ± SD were analyzed by One-way ANOVA of different groups and Dunn’s test for multiple group comparisons with three different experiments with three technical replicates from each experiments. (n = 9; *P < 0.05; **P < 0.001). Refer Fig. S9 for adhesion and intracellular survival of Pg strains in AEPE cells.

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