Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system.

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.

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.

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

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.

Clustering subspecies of Aeromonas salmonicida using IS630 typing.

BACKGROUND: The insertion element IS630 found in Aeromonas salmonicida belongs to the IS630-Tc1-mariner superfamily of transposons. It is present in multiple copies and represents approximately half of the IS present in the genome of A. salmonicida subsp. salmonicida A449. RESULTS: By using High Copy Number IS630 Restriction Fragment Length Polymorphism (HCN-IS630-RFLP), strains of various subspecies of Aeromonas salmonicida showed conserved or clustering patterns, thus allowing their differentiation from each other. Fingerprints of A. salmonicida subsp. salmonicida showed the highest homogeneity while 'atypical' A. salmonicida strains were more heterogeneous. IS630 typing also differentiated A. salmonicida from other Aeromonas species. The copy number of IS630 in Aeromonas salmonicida ranges from 8 to 35 and is much lower in other Aeromonas species. CONCLUSIONS: HCN-IS630-RFLP is a powerful tool for subtyping of A. salmonicida. The high stability of IS630 insertions in A. salmonicida subsp. salmonicida indicates that it might have played a role in pathoadaptation of A. salmonicida which has reached an optimal configuration in the highly virulent and specific fish pathogen A. salmonicida subsp. salmonicida.

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity.

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetem comitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A beta(2)-integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/ CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.

Probing of Actinobacillus pleuropneumoniae ApxIIIA toxin-dependent cytotoxicity towards mammalian peripheral blood mononucleated cells.

BACKGROUND: Actinobacillus pleuropneumoniae, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s) has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs) isolated from several species. FINDINGS: Toxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant. CONCLUSION: We have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues.

BACKGROUND: Lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX)-producing bacteria. RESULTS: The porcine-LFA-1 CD11a (alpha) subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant. CONCLUSION: Together with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.