Abstract
BackgroundAeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth.ResultsAmong the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized.ConclusionsIn this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.
Highlights
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide
The aim of this work was to use high-throughput proteomics to display the secretome of A. salmonicida subsp. salmonicida wild-type and an isogenic Type-three secretion system (T3SS)-deficient mutant (ΔascV, extremely lowvirulent) during the exponential-growth phase (GP) and stationary phase (SP)
Comprehensive analysis of the A. salmonicida secretome The extracellular proteins of exponential and stationary growth phase cultures of A. salmonicida were separated from bacterial pellets by centrifugation and concentrated from identical volumes of filtrated cell-free culture supernatant (SN) as described in Materials and Methods
Summary
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. Salmonicida, a Gramnegative bacterium, is the etiologic agent of furunculosis, a frequent and major pathogen of fisheries worldwide which is generating significant economic losses related to deficits in zootechnical profits and the intensive use virulence, as independent studies have shown that isogenic mutant strains for T3SS structural proteins are nonvirulent both in vitro and in vivo [4,5,6,7,8]. Results and discussions are separated into two parts In this first part, the authors focus on the general analysis of data, the discovery of cytoplasmic proteins with putative moonlighting activities in supernatants of A. salmonicida and the identification of putative antigens for fish vaccination against furunculosis. The authors characterize the whole in vitro repertoire of T3SS effectors and discuss the roles of the well-described and new putative virulence factors of A. salmonicida [11]
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