Host cell invasion and intracellular residence by Aeromonas salmonicida: role of the S-layer

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages. Here we report that S+ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants. When reconstituted with exogenous S-layers, these S- mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S+ and S- A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S+ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

Siderophore production by Aeromonas salmonicida.

Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), alpha, alpha'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. salmonicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophore-independent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.     

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.     

DNA probe for Aeromonas salmonicida.

A DNA fragment that is specific to Aeromonas salmonicida has been isolated from a genomic DNA library by differential hybridization. The specificity of this fragment as a DNA probe for A. salmonicida was shown by hybridization against reference strains and clinical isolates of A. salmonicida, related aeromonads, and species from several other bacterial genera. The sensitivity of detection by a polymerase chain reaction test, based on this fragment, was approximately two A. salmonicida cells.     

Pulsed-field gel electrophoriesis analyis of Aeromonas salmonicida ssp. salmonicida

A total of 133 strains of Aeromonas salmonicida ssp. salmonicida, isolated from a wide variety of sources, were characterized by pulsed-field gel electrophoresis patterns. Sixteen profiles were demonstrated, with one profile being predominant in samples from all the countries and species of fish. Our results suggest a clonal distribution of this subspecies, with a predominant clone being responsible for most of the outbreaks worldwide.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

Acquisition of iron by Aeromonas salmonicida.

The ability of six typical and three atypical strains of Aeromonas salmonicida to sequester Fe3+ from the high-affinity iron chelators ethylenediaminedihydroxy-phenylacetic acid, lactoferrin, and transferrin was determined. Typical strains were readily able to sequester Fe3+ and used two different mechanisms. One mechanism was inducible and appeared to involve production of a low-molecular-weight soluble siderophore(s). Iron uptake by this mechanism was strongly inhibited by ferricyanide. One virulent strain displayed a second mechanism which was constitutive and required cell contact with Fe3+-lactoferrin or -transferrin. This strain did not produce a soluble siderophore(s) but could utilize the siderophore(s) produced by the other strain. Fe3+ uptake by this stripping mechanism was strongly inhibited by dinitrophenol. Atypical strains displayed a markedly reduced ability to sequester iron from high-affinity chelators, although one of them was able to utilize the siderophores produced by the typical strain. In all strains examined, Fe3+ limitation resulted in the increased synthesis of several high-molecular-weight outer membrane proteins.     

Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.     

Evidence against dormacy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida

Results indicate that A. salmonicida does not enter an unculturable dormant state. The resuscitation of dormant cells by nutrient broth described by previous workers appears to be due to the presence of small numbers of viable, culturable cells too few to be detected by the sampling protocol employed.     

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.     

异育银鲫源杀鲑气单胞菌杀鲑亚种的分离鉴定

【目的】分离鉴定江苏省扬州市养殖场异育银鲫患病病原。【方法】采用常规的理化特性和分子生物学的方法,对从濒死异育银鲫肝脏处分离到的菌株YZ-1进行表型生物学、分子生物学及药敏试验的系统研究。【结果】该菌株16S r RNA基因(序列长度1 446 bp,Gen Bank登录号为JX164202)与其它杀鲑气单胞菌16S r RNA基因一致性在99%-100%之间,构建发育树确定该菌株为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicida subsp.salmonicida)。人工回感可导致异育银鲫死亡。药敏试验结果显示:对头孢呋辛、复方新诺明、恩诺沙星等23种抗生素敏感;对阿米卡星、四环素、大观霉素、头孢拉定等11种抗生素中度敏感;对青霉素G、链霉素、庆大霉素、氟苯尼考、万古霉素等10种抗生素耐药。【结论】研究结果证实引起异育银鲫死亡的病原为杀鲑气单胞菌杀鲑亚种。     

Interaction of Pseudomonas and Enterobacteriaceae plasmids in Aeromonas salmonicida.

We observed that Aeromonas salmonicida ARO200 will maintain either or both the Pseudomonas R-factor, pMG1, and Enterobacteriaceae R-factors. This bacterial strain, therefore, provides a unique background wherein the host ranges of Pseudomonas and Enterobacteriaceae plasmods overlap. Co-maintenance of these plasmids resulted in behavior of plasmid aggregates that allowed transfer of R-dterminants beyond the host range of the parent plasmid. We observed that the ARO200 genetic background facilitated the redistribution of B determinants among unrelated and conjugally noninterfertile gram-negative bacteria. Aberrant behavior resulting in the deletion of R-determinants for plasmids singly maintained in ARO200 was also observed. Plasmids studied included RP1, R702, IncP; Rs-a, IncW; R192.7, IncFII; R64-11, IncI; R390, IncN; and R6K, IncX.     

Survival of nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides of typical and atypical strains of the fish pathogen Aeromonas salmonicida. 32P intrinsically radiolabeled lipopolysaccharide in sarcosinate-extracted outer membrane preparations, lipopolysaccharide stained by silver in proteinase K-digested outer membrane preparations and whole cell lysates, as well as purified lipopolysaccharide, displayed O-polysaccharide chains which were unusually homogeneous with respect to chain length. Chemical analysis further revealed that the sugar composition of the smooth lipopolysaccharide purified from three typical strains was very similar. Immunoblotting and immunofluorescent staining with both polyclonal and monoclonal antibody showed that the O-polysaccharide chains were strongly immunogenic and were antigenically cross-reactive on typical and atypical strains from diverse origins. Immunofluorescence analysis and phage binding studies demonstrated that a number of these O-polysaccharide chains traversed the surface protein array of virulent strains of A. salmonicida and were exposed on the cell surface.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

Three small,cryptic plasmids from Aeromonas salmonicida subsp. salmonicida A449

The nucleotide sequences of three small (5.2-5.6 kb) plasmids from Aeromonas salmonicida subsp. salmonicida A449 are described. Two of the plasmids (pAsa1 and pAsa3) use a ColE2-type replication mechanism while the third (pAsa2) is a ColE1-type replicon. Insertions in the Rep protein and oriV region of the ColE2-type plasmids provide subtle differences that allow them to be maintained compatibly. All three plasmids carry genes for mobilization (mobABCD), but transfer genes are absent and are presumably provided in trans. Two of the plasmids, pAsa1 and pAsa3, carry toxin-antitoxin gene pairs, most probably to ensure plasmid stability. One open reading frame (ORF), orf1, is conserved in all three plasmids, while other ORFs are plasmid-specific. A survey of A. salmonicida strains indicates that pAsa1 and pAsa2 are present in all 12 strains investigated, while pAsa3 is present in 11 and a fourth plasmid, pAsal1, is present in 7.     

Inhibition of lipopolysaccharide synthesis in Agrobacterium tumefaciens and Aeromonas salmonicida.

Lipopolysaccharide (LPS) synthesis was inhibited, new lipid A metabolites accumulated, and growth ceased, when the plant pathogen Agrobacterium tumefaciens and the fish pathogen Aeromonas salmonicida were treated with an antibacterial agent which specifically inhibits CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthase). The new lipid A metabolites were purified by chromatography on DEAE-cellulose and chemically analysed. Metabolites isolated from both bacterial species contained glucosamine and phosphate in a 1:1 molar ratio, and 3-OH-C14:0 was the major fatty acid present (1 mol and 1.4 mol per mol glucosamine for A. tumefaciens and A. salmonicida, respectively). Inhibition of LPS synthesis by CMP-KDO synthase inhibitor had no effect on the initial kinetics of A. tumefaciens attachment to cultured carrot cells, but did inhibit cell aggregation normally induced by bacterial cellulose synthesis. Bacteria treated with inhibitor remained viable and able to synthesize protein at 15% the rate of control cells, indicating that the lack of cellulose-induced aggregation was not due to the inability of bacteria to make protein, but rather the inability to respond normally to the bacterial-plant cell interaction.