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Figure 4 | Veterinary Research

Figure 4

From: Orf1B controls secretion of T3SS proteins and contributes to Edwardsiella piscicida adhesion to epithelial cells

Figure 4

Orf1B contributes to E. piscicida adhesion to fish EPC cells by facilitating secretion of EseJ. A Representative images of infected EPC cells. Fish EPC cells were infected at an MOI of 10. After a 30-min incubation, cells were fixed and stained with anti-LPS (E. piscicida) antibody and Alex 488 Goat anti-mouse antibody before being imaged under confocal microscope. Bars, 50 μm. B Quantification of cell-associated bacteria per view. Fourteen views from each infection were quantified, and the mean ± SD from one representative experiment are shown. ***P < 0.001. C Increased steady-state protein level of EseJ upon disruption of Orf1B. Total bacterial proteins (TBP) and extracellular proteins (ECP) from similar amounts of WT strain, Δorf1B strain, and Δorf1B/orf1B strain cultured in TSB were probed with anti-EseJ and anti-EvpC antibodies. EvpC, a protein secreted by T6SS but not by T3SS, was used as a loading control. The immunoblotting shown are representative of three independent experiments. D The transcription level of eseJ was not interfered by the disruption of Orf1B. The mRNA levels of eseJ in wild-type strain, Δorf1B strain, and Δorf1B/orf1B strain were examined by qRT-PCR. Gene expression levels in the Δorf1B strain, and Δorf1B/orf1B strain relative to the level in the wild-type strain are presented as relative fold changes in gene expression. 16S rRNA was used as the reference gene. Data are presented as means ± SD. The t test implemented in SPSS was used to calculate the P value with a hypothetical mean of 1.0. NS, not significantly different.

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