The Aeromonas salmonicida subsp. salmonicida exoproteome: global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis

BACKGROUND Aeromonas 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. RESULTS Among 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. CONCLUSIONS In 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