Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.

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.

AopP, a type III effector protein of Aeromonas salmonicida, inhibits the NF-kappaB signalling pathway

Aeromonas salmonicida subsp. salmonicida contains a functional type III secretion system that is responsible for the secretion of the ADP-ribosylating toxin AexT. In this study, the authors identified AopP as a second effector protein secreted by this system. The aopP gene was detected in both typical and atypical A. salmonicida isolates and was found to be encoded on a small plasmid of approximately 6.4 kb. Sequence analysis indicates that AopP is a member of the YopJ family of effector proteins, a group of proteins that interfere with mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa B (NF-kappaB) signalling pathways. AopP inhibits the NF-kappaB pathway downstream of IkappaB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Alternative host model to evaluate Aeromonas virulence

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.

Pseudomonas chlororaphis strain JF3835 reduces mortality of juvenile perch, Perca fluviatilis L., caused by Aeromonas sobria

Motile aeromonad septicaemia caused by Aeromonas sobria is a cause of disease in farmed perch, Perca fluviatilis L., in Switzerland. We have evaluated the potential of a Pseudomonas chlororaphis isolate, obtained from perch intestine, to control A. sobria infection. Inoculation of juvenile perch with P. chlororaphis strain JF3835 prior to infection with A. sobria caused a reduction in A. sobria associated mortalities. Infection of perch with xylE-labelled P. chlororaphis indicated the bacterium is able to transiently colonize juvenile fish and fingerlings.

Outbreaks of an ulcerative and haemorrhagic disease in Arctic char Salvelinus alpinus caused by Aeromonas salmonicida subsp. smithia

Arctic char Salvelinus alpinus farmed in different places in Austria and free of the viral diseases viral haemorrhagic septcaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) experienced disease and mortality. Diseased fish showed skin ulceration and pathological signs of sepsis. Aeromonas sp. was isolated as pure culture from the kidney of freshly euthanized diseased fish. Three independent isolates from outbreaks that occurred on 2 of the affected farms were analyzed phylogenetically by DNA sequence analysis of the rrs and gyrB genes and phenotypically with biochemical reactions. All 3 isolates were identified as Aeromonas salmonicida subsp. smithia. Analysis of virulence genes in these isolates revealed the presence of a Type III secretion system as well as several related virulence effector genes including aexT, encoding the Aeromonas exotoxin AexT, aopP and aopH. These genes are characteristic for virulent strains of typical and atypical subspecies of A. salmonicida.

Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences

Genetic relationships among bacterial strains belonging to the genus Aeromonas were inferred from 16S rRNA, gyrB and rpoB gene sequences. Twenty-eight type or collection strains of the recognized species or subspecies and 33 Aeromonas strains isolated from human and animal specimens as well as from environmental samples were included in the study. As reported previously, the 16S rRNA gene sequence is highly conserved within the genus Aeromonas, having only limited resolution for this very tight group of species. Analysis of a 1.1 kb gyrB sequence confirmed that this gene has high resolving power, with maximal interspecies divergence of 15.2 %. Similar results were obtained by sequencing only 517 bp of the rpoB gene, which showed maximal interspecies divergence of 13 %. The topologies of the gyrB- and rpoB-derived trees were similar. The results confirm the close relationship of species within the genus Aeromonas and show that a phylogenetic approach including several genes is suitable for improving the complicated taxonomy of the genus.

Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida subsp. salmonicida

An ADP-ribosylating toxin named Aeromonas salmonicida exoenzyme T (AexT) in A. salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, was characterized. Gene aexT, encoding toxin AexT, was cloned and characterized by sequence analysis. AexT shows significant sequence similarity to the ExoS and ExoT exotoxins of Pseudomonas aeruginosa and to the YopE cytotoxin of different Yersinia species. The aexT gene was detected in all of the 12 A. salmonicida subsp. salmonicida strains tested but was absent from all other Aeromonas species. Recombinant AexT produced in Escherichia coli possesses enzymatic ADP-ribosyltransferase activity. Monospecific polyclonal antibodies directed against purified recombinant AexT detected the toxin produced by A. salmonicida subsp. salmonicida and cross-reacted with ExoS and ExoT of P. aeruginosa. AexT toxin could be detected in a wild type (wt) strain of A. salmonicida subsp. salmonicida freshly isolated from a fish with furunculosis; however, its expression required contact with RTG-2 rainbow trout gonad cells. Under these conditions, the AexT protein was found to be intracellular or tightly cell associated. No AexT was found when A. salmonicida subsp. salmonicida was incubated in cell culture medium in the absence of RTG-2 cells. Upon infection with wt A. salmonicida subsp. salmonicida, the fish gonad RTG-2 cells rapidly underwent significant morphological changes. These changes were demonstrated to constitute cell rounding, which accompanied induction of production of AexT and which led to cell lysis after extended incubation. An aexT mutant which was constructed from the wt strain with an insertionally inactivated aexT gene by allelic exchange had no toxic effect on RTG-2 cells and was devoid of AexT production. Hence AexT is directly involved in the toxicity of A. salmonicida subsp. salmonicida for RTG-2 fish cells.