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. 2013;8(1):e54934.
doi: 10.1371/journal.pone.0054934. Epub 2013 Jan 28.

Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis

Fernando A Gómez  1 , Jaime A Tobar, Vitalia Henríquez, Mariel Sola, Claudia Altamirano, Sergio H Marshall
Affiliations

Evidence of the presence of a functional Dot/Icm type IV-B secretion system in the fish bacterial pathogen Piscirickettsia salmonis

Fernando A Gómez et al. PLoS One. 2013.

Abstract

Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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
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
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.
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This work was supported by FONDECYT grant 1120584 for Sergio H. Marshall and by a Conicyt Doctoral Scholarship for Fernando A. Gómez. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.