Differential partition of virulent Aeromonas salmonicida and attenuated derivatives possessing specific cell surface alterations in polymer aqueous-phase systems

Two-polymer aqueous-phase systems were used to compare via partitioning the surface properties of strains of the fish pathogen Aeromonas salmonicida which differed in their ability to produce the surface protein array known as the A layer and in their ability to produce smooth lipopolysaccharide. In these two-phase systems, biological particles are known to partition between the phases in a manner related to a variety of surface properties, including hydrophobicity, charge, and lipid composition. Both the presence of the superficial protein layer and the O polysaccharide chains of lipopolysaccharide were shown to play an important role in the partitioning behavior of A. salmonicida cells. The presence of the A layer, which is crucial to the virulence of A. salmonicida, appeared to decrease the surface hydrophilicity of this pathogen and to increase, in a somewhat specific manner, its surface affinity for fatty acid esters of polyethylene glycol. The ability of two-polymer aqueous-phase systems to differentially partition A. salmonicida cells on the basis of differences in surface architecture suggests their general usefulness for the analysis of surface properties important in bacterial virulence and should permit their use in the selection of strains and mutants exhibiting specific surface characteristics.     

Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Electrostatic mechanism of survival of virulent Aeromonas salmonicida strains in river water.

The ecological mechanism of survival of Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, in river water was investigated by laboratory-based experiments with two virulent strains (which were autoagglutinating) and two virulent strains (which were nonagglutinating). A difference in net electrical charge of A. salmonicida cells was detected by electrophoresis; cells of the virulent strains were negative, whereas cells of the avirulent strains were positive. Despite the loss of viable cells within a week in distilled water and physiological saline (0.85% sodium chloride), the cells of the virulent strains survived for more than 15 weeks in the presence of diluted humic acid (10 micrograms/ml), tryptone (10 micrograms/ml), and cleaned river sand (100 g/100 ml of medium), but loss of viable cells occurred within 5 weeks in the absence of sand. The cells of the avirulent strains lost viability within 2 weeks with no relation to the presence of sand. Using ion-exchange columns, humic acid and the amino acids of tryptone were found to be anionic and cationic in water (pH 7.0), respectively. Sand particles had a high capacity to adsorb humic acid alone and amino acid-humic acid complexes. Thirty to fifty times the environmental concentration of amino acids (10 micrograms/ml) were accumulated on the surface of sand particles, thereby permitting only bacterial cells carrying net negative electrical charges (virulent cells) to survive for a long period on the surface of the sand particles. These electrostatic interrelationships among river sand, humic acid, and bacterial cells are closely implicated in the mechanism of long-term survival of virulent A. salmonicida in river sediments.     

A temperate bacteriophage specific for strains ofAeromonas salmonicida possessing A-layer,a cell surface virulence factor

A temperate bacteriophage designated TP446 was isolated from culture supernatants ofAeromonas salmonicida strain A446. Phage TP446 adsorbed to all of the typical and atypical strains ofA. salmonicida tested that possessed A-layer, the surface protein array that represents the primary virulence factor of this fish pathogen. In contrast, TP446 failed to adsorb to mutants lacking A-layer. These results indicate that the A-layer is a component of the receptor for phage TP446.     

Molecular characterization and quantitative analysis of superoxide dismutases in virulent and avirulent strains of Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), sodA and sodB. The sodA gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O(2)(-)-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative sodA were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of A. salmonicida subsp. salmonicida strain A449 when cultured in vitro and in vivo.     

Increased cell surface hydrophobicity associated with possession of an additional surface protein by Aeromonas salmonicida

Abstract The cell surface of strains of Aeromonas salmonicida possessing an additional surface protein (A-protein) was shown to be more hydrophobic than strains devoid of this protein, using the techniques of phase partitioning, agglutination in the presence of ammonium sulphate and hydrophobic interaction chromatography.     

Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida.

The fish pathogen Aeromonas salmonicida possesses a regular surface layer (or A-layer) which is an important virulence determinant. The A-protein, a single bilobed protein organized in a p4 lattice of M4C4 arrangement with two morphological domains, comprises this layer. The role of divalent cations in the A-layer structure was studied to better understand A-protein subunit interactions affecting structural flexibility and function. Divalent cation bridges were found to be involved in the integrity of the A-layer. Two novel A-layer patterns were formed as the result of growth under calcium limitation or by chelation of divalent cations with EDTA or EGTA, thereby constituting the first reported case of formation of distinct regular arrays upon divalent cation depletion. Furthermore, under these conditions A-protein was sometimes released as tetrameric units, rather than in monomeric form. The formation of the two novel patterns is best explained by a sequence of structural rearrangements, following disruption of only one of the two A-layer morphological units, that is, those held together by divalent cation bridges. The free tetrameric units represent four A-protein subunits clustered around the unaffected four-fold axis.     

Effects of nutrients on exopolysaccharide production and surface properties of Aeromonas salmonicida.

Extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Aeromonas salmonicida A450 and the influence of the capsule on cell surface properties were studied. A. salmonicida did not produce CPS or EPS when glucose, phosphate, magnesium chloride, or trace mineral components were absent from the medium. The addition of yeast extract improved capsule production. Neither EPS nor CPS formation depended on the C/N ratio, although it appeared to be influenced by the level of carbon and nitrogen in the culture. Both EPS and CPS production started at the end of the logarithmic growth phase. The amounts of EPS and CPS produced were not influenced by temperature changes between 15 and 20 degrees C and was maximal from pH 7 to 7.5. Cell surface properties were strongly influenced by capsule production; high CPS production was associated with enhanced cell hydrophilicity and autoagglutination. The effect of CPS on cell surface properties was independent of the presence of the surface protein array (A-layer).     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida

The identification of lipopolysaccharide as periodic acid-Schiff positive material, present in the membrane fraction of the fish pathogenic Gram-negative bacterium Aeromonas salmonicida, analyzed by SDS-polyacrylamide gel electrophoresis, is shown. Such analysis has revealed several periodic acid-Schiff positive bands and many membrane proteins among which a pathogenicity-related Mr 54000 protein as a constituent of an additional surface layer outside the outer membrane (Evenberg et al., (1982) Biochim. Biophys. Acta 684, 241-248). The latter protein, designated as additional cell envelope protein or ACE protein, has been purified and characterized in our laboratory (Evenberg and Lugtenberg, (1982) Biochim. Biophys. Acta 684, 249-254). Most strains produce both high and low molecular weight lipopolysaccharide species, presumably corresponding with the presence and (virtual) absence, respectively, of an O-antigenic chain. The property to produce high molecular weight lipopolysaccharide can be lost upon subculturing in laboratory growth media and such is greatly enhanced by the prior loss of the ability to produce ACE protein. Lipopolysaccharide and ACE protein were identified as the major antigens. A new polysaccharide-like antigen, designated as PS-antigen, was detected. Moreover, immunological indications for the presence of a lipoprotein in A. salmonicida are described. The surface localization of the antigens was determined by testing whether preadsorption of antisera by intact cells decreased the binding of IgG to these antigens, or decreased the ability of the sera to agglutinate cells. According to these criteria lipopolysaccharide, ACE protein and PS-antigen are the major surface-located antigens. Material cross-reactive with lipopolysaccharide, ACE protein and PS-antigen has been found in a large number of strains. Several lines of evidence indicate the presence of interactions between ACE protein and lipopolysaccharide. Based on these results a molecular model of the cell envelope of virulent A. salmonicida is presented.     

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from typical and atypical strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.Abbreviations A-layer additional surface protein layer - A-protein surface protein - Ast Aeromonas salmonicida typical - Asa Aeromonas salmonicida atypical - A^- phenotypically A-protein-negative variant - O^- phenotypically O-polysaccharide-negative variant - O^wk phenotypically O-polysaccharide weak variant - BHI brain heart infusion - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TEM transmission electron microscopy     

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.     

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.     

Physical and functional S-layer reconstitution in Aeromonas salmonicida.

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.     

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.     

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.     

Differential immune response of adipocytes to virulent and attenuated Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tb) takes advantage of various cell types, allowing it to remain in the host for long periods. Because adipocytes have been proposed as niches for dormant M. tb in the latent state, understanding the interaction of virulent M. tb with adipocytes is important. We compared changes in cytokine secretion from 3T3-L1 murine adipocytes infected with virulent M. tb H37Rv (V-M. tb) and attenuated M. tb H37Ra (A-M. tb) strains. Both strains maintained non-replicating states within adipocytes until 10 days post-infection. Adipocytes infected with V-M. tb secreted lower levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12p40, IL-6, and IL-17, and lower levels of nitric oxide than those infected with A-M. tb. In contrast, the anti-inflammatory cytokines, IL-10 and IL-4, were markedly induced in V-M. tb-infected adipocytes versus those infected with A-M. tb at an early time point. Heat-killed or formalin-fixed bacteria induced lower levels of cytokines and no difference was observed between strains. Moreover, V-M. tb induced a high level of necrosis versus A-M. tb in conjunction with increased levels of LHD. These results suggest that V-M. tb regulates cytokine expression in its favor, increasing cytokines necessary for immune evasion and decreasing those required for protective immunity.     

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.     

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.