Identification and characterization of virulence effectors Dot/Icm-related in the fish bacterial pathogen Piscirickettsia salmonis Arredondo-Zelada, Oscar1., Flores-Herrera, Patricio1., Henríquez, Fabián A.1., Marshall, Sergio H.1., Gómez, Fernando A.1. 1
Laboratorio de Genética e Inmunología :Molecular, Ciencias, Pontificia Universidad Católica de Valparaíso.
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Introducction
Metodology
Piscirickettsia salmonis is a gramnegative bacterium, etiological agent of Piscirickettsiosis, an infectious disease that affects the global salmon farming since the late 80’s. P. salmonis is facultative intracellular organism, not mobile and is able to infect and survive in fish macrophages. Recent reports confirm that this agent have a Dot/Icm Secretion System, homologue to the described for Legionella pneumophila and Coxiella burnetii. This work focused in make an in silico and molecular characterization of putative virulent effectors (Dot/Icm substrates) in P. salmonis, using as reference those described for L. pneumophila and C. burnetii. To do so, 294 reference Dot/Icm substrates of L. pneumophila and C. burnetii were screened in the genome of P. salmonis LF-89, using a local BLASTP of the RAST server in order to find homologies. Was possible identify 5 proteins of P. salmonis with homologies to the reference Dot/Icm substrates, which contains eukaryotic conserved domains (Serine-threonine kinase C, SEL-1, U-Box and ankyrins repeats), a typical feature of Dot/Icmrelated virulence effectors.
Funding Supported by the grant FONDECYT N° 11130407
Table 1: Dot/Icm Secretion signal present on P. Salmonis Dot/Icm Substrates. Hypothetical sequences selection RAST
Blast against P. salmonis Genome
Extract from NCBI protein sequences Putative Candidates Obtained belonging to potentials virulence effectors L. pneumophila and C. Burnetti virulence referenced as effectors
Figure 1: 3-D structural modelling of the putative P. salmonis Dot/Icm Substrates and the respective C-terminus region. All models were selected from Raptor X and Phyre2 servers, because present the higher confidence levels by Qmean and ModFolds analyses.
Ank-A SMART CDD MOTIF PKc-1
Sel-1 Candidate sequences with domains characterized Modeling of candidate sequences
Additionally, the 5 P. salmonis proteins contain the classic Dot/Icm secretion signal in the C-terminal (E-Block and hydrophobic or proline residue at position analyzed -3 or -4). Finally, the nucleotide sequence of the five Dot/Icm substrates were cloned into pYES3-CT vector and the proteins will be expressed in Saccharomyces cerevisiae, in order to evaluate their effect over eukaryotic cells.
Results
select sequences according to E-value
Analysis candidate sequences (C-terminal signal)
RAPTORX
PHYRE2
Ubox-1
Sequences cloned in expression vector for S. cerevisiae
Unk-1
Projections Experimental validation of all bioinformatics analysis, in order to confirm that these proteins corresponding to real virulence effector secreted by the Dot/Icm system in P. salmonis: 1. To evaluate the effect of the putative P. salmonis virulence effector over eukaryotic cells, using S. cerevisiae as model. 2. Analyze the expression level of the putative Dot/Icm substrate in different growth conditions (infection in macrophages cell lines and in cell-free medium). 3. Predict the putative function/role of the Dot/Icm substrates during the infection, using in silico approaches.
