The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

Abstract

Background: Aeromonas 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.

Results: Results 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.

Conclusions: We 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.

Figure 1

SDS gel electrophoresis of A. salmonicida subsp. salmonicida proteins from GP wt andΔascV mutant SNs stained with Coomassie blue. SDS gel electrophoresis of proteins from supernatants (SN) of wild-type (wt) and ΔascV mutant (mut) strains in exponential (GP) phase of growth. Proteins corresponding to the most abundant bands are indicated. The molecular weights (kDa) of the Protein Ladder are shown on both sides of the figure.

Figure 2

Ratios of different T3SS components in A. salmonicida wt versus mutant strain. The plot represents the logarithm base two of the ratios [wt/mut] PMSS values for each protein identified in pellet (X-axis) and SNs (Y-axis). For T3SS components, exponential growth phase (GP) values are represented by circles and stationary phase (SP) values by squares. Red, the T3SS effectors; dark green, T3SS chaperones; white, translocators; black, needle components; orange, OM secretin ring; light green, IM export ring; dark blue, C ring ATPase; light blue, transcriptional regulators. Values of other proteins in GP and SP are shown with dark blue triangles and yellow squares respectively. The hatched line represents the threshold that we used to identify proteins 4-fold more present in wt SNs.

Figure 3

Proportion of T3SS effectors and translocators. The total PMSS value of T3SS effectors or translocators present in wt and mutant SNs during GP and SP are shown in diagram (A). Quantities were strongly decreased in mutant SNs but low levels of T3SS effectors/translocators were detected and increased during SP. Diagram (B), represents the proportion of [extra- or intrabacterial effectors or translocators/T3SS structural components] calculated with the total PMSS value of each category. These results showed that despite the ascV deletion, A. salmonicida kept the same proportion between intrabacterial effectors/translocators and the other components of the T3SS while strong differences were observed between wt and mutant for the proportion of extrabacterial effectors/translocators.

Figure 4

Protein abundance of A. salmonicida appendages. The diagram represents total PMSS values of A. salmonicida secretion systems (T1- to T6SS), flagella (lateral and polar), pili (T1 pilus [Fim], T4 pili [Tap, Flp and Msh] and fimbriae (MatB). Only the structural components were taken into account for the T3SS. Logically, the strongest difference in protein amount was observed for the T3SS. No differences were observed in pellets for T1- (103 ABC-transporters) and T2SS. In SNs, the strong difference between wt and mutant strains for T1SS values is due to the higher amount of VapA in mutant SNs.

Figure 5

Correlation of protein contents between wt and T3SS-mutant. Each plot represents the PMSS (A and B) or LFQ (C and D) values for each protein identified in wt (X-axis) and/or mutant (Y-axis) strains, in supernatants (SNs, A and C) and pellets (B and D). Values of exponential growth phase (GP) are dark blue triangles and stationary phase (SP) values are yellow squares. The global distribution of wt vs mutant protein values was linear in all conditions, but with a larger repartition in SNs than in pellets, thereby indicating differences in protein secretion between wt and ΔascV strains. Red values = T3SS proteins; orange = VapA, AerA, AerB, Ahe2, SatA and Asx; light blue = TagA, microbial collagenase, extracellular desoxyribonuclease ASA_1199, PlaA1, PlaC, Amy1, CdxA, ChiB, Chi2 and AmyA.