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Gomez F.A.,Pontifical Catholic University of Valparaiso | Tobar J.A.,Laboratorio Centrovet | Henriquez V.,Pontifical Catholic University of Valparaiso | Sola M.,Laboratorio Centrovet | And 3 more authors.
PLoS ONE | Year: 2013

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. © 2013 Gómez et al. Source

Labra A.,Pontifical Catholic University of Valparaiso | Arredondo-Zelada O.,Pontifical Catholic University of Valparaiso | Flores-Herrera P.,Pontifical Catholic University of Valparaiso | Marshall S.H.,Pontifical Catholic University of Valparaiso | And 2 more authors.
Microbial Pathogenesis | Year: 2016

Piscirickettsia salmonis seriously affects the Chilean salmon industry. The bacterium is phylogenetically related to Legionella pneumophila and Coxiella burnetii, sharing a Dot/Icm secretion system with them. Although it is well documented that L. pneumophila and C. burnetii secrete different virulence effectors via this Dot/Icm system in order to attenuate host cell responses, to date there have been no reported virulence effectors secreted by the Dot/Icm system of P. salmonis. Using several annotations of P. salmonis genome, here we report an in silico analyses of 4 putative Dot/Icm effectors. Three of them contain ankyrin repeat domains and the typical conserved 3D structures of this protein family. The fourth one is highly similar to one of the Dot/Icm-dependent effectors of L. pneumophila. Additionally, all the potential P. salmonis effectors contain a classical Dot/Icm secretion signal in their C-terminus, consisting of: an E-Block, a hydrophobic residue in -3 or -4 and an electronegative charge. Finally, qPCR analysis demonstrated that these proteins are overexpressed early in infection, perhaps contributing to the generation of a replicative vacuole, a key step in the neutralizing strategy proposed for the Dot/Icm system. In summary, this report identifies four Dot/Icm-dependent effectors in P. salmonis. © 2015 Elsevier Ltd. Source

Marshall S.H.,Pontifical Catholic University of Valparaiso | Marshall S.H.,Fraunhofer Chile Research Foundation | Gomez F.A.,Pontifical Catholic University of Valparaiso | Ramirez R.,Pontifical Catholic University of Valparaiso | And 3 more authors.
Research in Microbiology | Year: 2012

Piscirickettsia salmonis is a bacterial fish pathogen seriously threatening the sustainability of the Chilean salmon industry. The biology and life cycle of this bacterium is not completely understood and there are no reports explaining how it survives or persists in marine environments. This work provides descriptive data of P. salmonis behavior when it is exposed to stress conditions, producing large cell aggregates closely resembling typical biofilm structures. In order to track this putative biofilm, we used indirect fluorescence and scanning electron microscopy. Complex masses were observed over time; the bacteria appear to be embedded within a matrix which disappears when it is exposed to cellulase, suggesting a polysaccharide nature typical of biofilm formation. Two lectins (ConA and WGA) were used to characterize the matrix. Both lectins showed a strong reaction with the structure, validating the exopolysaccharide nature of the matrix. Recently, several studies have demonstrated a correlation between toxin/anti-toxin system expression at initial stages of biofilm formation. In this report, QRT-PCR analysis was used with the P. salmonis toxin/anti-toxin mazEF operon, showing induction of these genes at early stages of biofilm formation, suggesting that said formation may be an adaptive strategy for survival and persistence under stress conditions in marine environments. © 2012 Institut Pasteur. Source

Ramirez R.,Fraunhofer Chile Research Foundation | Gomez F.A.,Pontifical Catholic University of Valparaiso | Marshall S.H.,Pontifical Catholic University of Valparaiso | Marshall S.H.,Fraunhofer Chile Research Foundation
FEMS Microbiology Letters | Year: 2015

Piscirickettsia salmonis is an aggressive fish pathogen that causes Piscirickettsiosis, a systemic disease that threatens the sustainability of salmon production in Chile. To date, the infection strategies of this bacterium are poorly characterized, a Dot/Icm Type IV Secretion System homolog for intracellular multiplication and survival in macrophages is suggested. Since an invading pathogen and its host develop a complex interaction in which the pathogen strives to survive and replicate, while the host tries to eliminate infected cells and the invading pathogen, we decided to evaluate how the bacterium enters macrophages, its preferred target in vivo, and to follow its fate while struggling with its host using actin cytoskeleton as a molecular marker. We were able to demonstrate that clathrin is required for internalization and that actin cytoskeleton plays a demonstrative role throughout the infective process. Indeed, unlike other fish pathogens, P. salmonis fully exploits the actin monomers both from the disorganized cytoskeleton and an apparently pathogen-induced de novo synthesis of actin, generating tridimensional vacuoles that are increasingly detected at later stages of infection. We expect our results to contribute to a better understanding of the pathogenesis of this important fish pathogen. © FEMS 2014. Source

Diaz C.,Fraunhofer Institute for Molecular Biology and Applied Ecology | Laurie V.F.,University of Talca | Laurie V.F.,Fraunhofer Chile Research Foundation | Molina A.M.,San Sebastian University | And 2 more authors.
American Journal of Enology and Viticulture | Year: 2013

The organic and mineral components of 20 qvevri wines from selected wineries throughout Europe were evaluated to gain insight into one of the oldest known methods of wine production. The pH, residual glucose, titratable acidity, specific organic acids (acetic, lactic, malic, and tartaric acids), phenolics (total phenolics and anthocyanins in red wines), total antioxidants, and selected minerals (phosphorus, calcium, magnesium, manganese, potassium and zinc) of the samples were measured. The data were compared with previously reported values for conventional wines. Qvevri wines were less acidic than conventional wines, containing higher levels of acetic and lactic acids but lower levels of tartaric acid. They also contained higher levels of antioxidants and total phenolics, the latter up to 10 times more abundant in the white wine varieties than in conventional wines. Despite fermentation in clay vessels, the mineral content of qvevri wines was within the normal reported range, although phosphorus levels were slightly higher. This is the only study that has considered the compositional nature of qvevri wines. © 2013 by the American Society for Enology and Viticulture. All rights reserved. Source

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