Abstract
BackgroundAeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth.ResultsResults confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected.ConclusionsWe demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.
Highlights
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide
We identified a total of 2136 A. salmonicida proteins with peptidematching score summation (PMSS) and label-free quantitation (LFQ) values among the different experimental conditions for 1861 and 2070 proteins respectively
The comparison by high-throughput proteomics of A. salmonicida secretomes from wt and Typethree secretion system (T3SS)-deficient strains is a powerful method that gave us the opportunity (i) to characterize the full in vitro repertoire of T3SS effectors represented mainly by AopH, Ati2, AexT, AopP, AopO, AopN and ExsE, (ii) to identify new putative virulence factors that are secreted in the extracellular medium or might be translocated into the host cell by the T3SS or alternative mechanisms, and (iii) to confirm that A. salmonicida secreted toxins, adhesins and enzymes that have been described until now and are found in this study are secreted to a higher extent in the extremely low-virulent ΔascV mutant
Summary
Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. 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 of antibiotics [1]. The T3SS is the only one recognized as having a major effect on virulence, as independent studies have shown that isogenic mutant strains for T3SS structural proteins are non-virulent both in vitro and in vivo [2,13,14,15,16]. Once injected in the eukaryotic cytosol, effector proteins are able to modulate cell signalling pathways, or alternatively disrupt the dynamics of the cytoskeleton, thereby modulating host cell biology for the benefit of the pathogen [17]
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