Table 1.
Degenerate primers used for the initial amplification of P. salmonis dotB, dotA and icmK genes.
Table 2.
Specific primers for P. salmonis dot/icm genes used for qRT-PCR.
Figure 1.
ClustalW alignments among the most conserved regions of P. salmonis dot/icm protein products with their homologues. A:
DotB alignment, where the black and red asterisks show the Walker A and B, respectively (ATP binding site); B: DotA alignment; C: IcmK alignment; D: IcmE alignment. All figures were created using Jalview, where the color intensity shows the conservation degree of the aminoacids between the sequences.
Table 3.
BLASTX results for P. salmonis dotB, dotA, icmK and icmE genes.
Table 4.
Hydrophobic domains of the P. salmonis DotA protein obtained using the SOSUI software.
Figure 2.
Expression profile of P. salmonis dot/icm genes during RTS11 and Sf21cell lines kinetic infection.
Gene expression was determined by qRT-PCR, using relative quantification. A: dotB gene expression number; B: icmE gene expression; C: icmK mRNA gene expression; D: dotA gene expression. Gene expression was normalized by the use of ITS like a housekeeping gene. 24 hours post-infection in each cell line was used as calibrator (value = 1).
Figure 3.
Expression profiles of dot/icm genes throughout P. salmonis growth kinetics at different pHs in MC1 medium.
Gene expression was determined by qRT-PCR, using relative quantification. A: dotB gene expression number; B: icmE gene expression; C: icmK gene expression; D: dotA gene expression. Gene expression was normalized by the use of ITS like a housekeeping gene. Two hours of growth at pH 7.0 was used as calibrator (value = 1) for all genes. The figure shows that all genes were notably over-expressed at pH 4.0, particularly at 2 hours of incubation.
Figure 4.
Confocal Laser Scanning Microscopy of P. salmonis infection on three cell lines showing the escape of phagosome-lysosome fusion.
The immunofluorescence was made 5 days post-infection. Lysosomes were stained in red with LysoTracker Red reagent and P. salmonis was detected with a FITC conjugated antibody. A: CHSE-214 cell line infected with P. salmonis. B: Sf21 cell line infected with P. salmonis. C: RTS11 cell line infected with P. salmonis. D: CHSE-214 cell line infected with formaldehyde-inactivated P. salmonis, the immunofluorescence stain was made at 48 hours after infection.