Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: II. Purification and characterization of a major cell envelope protein related to autoagglutination,adhesion and virulence

The purification of the major protein of the membrane fraction of an autoagglutinating strain of Aeromonas salmonicida is described. This protein, designated as additional cell envelope protein, is water-insoluble, has a molecular weight of about 54 000 and its amino terminal sequence is H₂N-Asp-Val-Leu-Leu. Neither sulphur-containing amino acids nor sugar residues were detected. Its amino acid composition, which shows that the additional cell envelope protein is hydrophobic in nature, is remarkably similar to those of various proteins known to be present in additional surface layers of other bacteria, to the adhesive K88 fimbriae of enteropathogenic Escherichia coli and to a pore protein of the outer membrane of E. coli K12.     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: I. Relationship between autoagglutination and the presence of a major cell envelope protein

A comparison was made of membrane protein patterns of various Aeromonas salmonicida strains, initially isolated from different habitats with respect to fish species affected, pathological entity, and geographic location of the outbreak of the disease. A major protein with a molecular weight of 54 000 was found in all autoagglutinating strains, whereas this protein is present in low amounts, or not at all, in non-autoagglutinating strains. Evidence for a causal relationship between the presence of this protein and the phenomenon of autoagglutination came from the observation that a change of the growth medium led simultaneously to an almost complete loss of the additional cell envelope protein and the property of autoagglutination. As it has already been reported that autoagglutination is correlated with the presence of an additional cell surface layer, we hypothesize that the additional cell envelope protein is the (major) subunit of this layer. The application of the gel immuno radio assay, an immunological technique suited to detect antigens in a gel, revealed that the additional cell envelope proteins of all tested strains are immunologically related. The possibility to the use of this protein as a component of a vaccine against A. salmonicida infections is discussed.     

A stable L-form of the fish pathogen Aeromonas salmonicida

A stable L-form of Aeromonas salmonicida , which resulted from induction with benzylpenicillin, grew on brain heart infusion agar at 0–5°C. The L-form was stored successfully for 10 months in phosphate-buffered saline supplemented with 150 g l^-1 glycerol at -70°C. Reversion of the L-form to parental-type walled cells was achieved by transfer to brain heart infusion broth with incubation at 25°C.     

Survival of the fish pathogen Aeromonas salmonicida in seawater

Survival of Aeromonas salmonicida in natural (non-sterile) seawater, as determined from colony counts on marine agar, was found to be influenced by the presence of potentially inhibitory organisms, i.e., Acinetobacter, Aeromonas hydrophila, Chromobacterium, Escherichia coli, Flavobacterium and Pseudomonas, and their metabolites. Yet, samples, thought to be devoid of culturable A. salmonicida, were found to contain cells, which were filterable through 0.22 and 0.45 microns Millipore Millex porosity filters, and were recoverable on a specialised medium for L-forms, i.e. L-F medium.     

Comparison of the surface properties of seven strains of a fish pathogen, Aeromonas salmonicida

Several physical properties related to the surface characteristics of autoaggregating and non-autoaggregating strains of Aeromonas salmonicida have been investigated. Properties examined included resistance to the bactericidal action of trout serum, adhesion to fish leucocytes and fish cell monolayers in vitro , resistance to phagocytosis by fish leucocytes and the in vivo localization following intraperitoneal injection. For each strain the presence or absence of an extracellular protein A-layer was investigated and the pathogenicity for brook trout determined.
Presumptive A-layer protein, in the form of a 49 kdal subunit, could be detected only in one of the strains examined. This strain autoaggregated and was the most resistant to serum bactericidal activity. Complement activation by the alternative pathway was thought to be responsible for this heat-labile bactericidal activity. Three strains that autoaggregated and three that did not had no detectable A-layer. Autoaggregating strains appeared more adhesive to both fish cell types but all strains were phagocytosed by fish leucocytes to a similar degree. An autoaggregating strain was localized in the spleen. The seven strains were only moderately pathogenic for brook trout, possibly as a result of the challenge system. In view of this, no property investigated could be correlated with greatly increased virulence.     

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.     

Starvation survival of the fish pathogen Aeromonas salmonicida in seawater

Abstract Three strains of the fish pathogenic bacterium Aeromonas salmonicida were examined with respect to their ability to survive in seawater. Five to seven days after plate counts decreased below the detection limit of 10 cells/ml, the population of respiring A. salmonicida cells comprised less than 4% of the initial total bacterial population. At this stage, samples were transferred either to sterile nutrient or to control flasks containing sterile nutrient-free medium. The addition of nutrients caused reappearance of growing bacteria, detected by plating on agar medium and an increase of the population of respiring bacteria to 85–95% of the total population. In a separate experiment it was shown that after more than six months of starvation at 15°C, a few of the starved A. salmonicida cells were able to regrow in liquid media after addition of nutrients, but not on agar media. These cells evade detection by direct microscopic respiratory measurement but appear to be reactivated after addition of nutrients.     

Dormant/unculturable cells of the fish pathogen Aeromonas salmonicida

Viable cells of Aeromonas salmonicida remained in experimental marine systems after plate counts indicated an absence of culturable cells. These so-called viable but nonculturable (VBNC) cells were coccoid and smaller than their normal culturable counterparts. There was no reduction in lipopolysaccharide of the VBNC cells. There was an alteration in protein composition, however, with a decline in some (15, 70, 30, 22, and 17 kDa), but an increase in another protein (49 kDa). A significant loss of DNA occurred. The VBNC cells responded to fluorescent antibodies prepared against A. salmonicida by developing enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), and they demonstrated respiratory activity. It was concluded that A. salmonicida survived in seawater, but major morphological changes occurred with cells retaining some viability but losing pathogenicity to Atlantic salmon (Salmo salar).     

Structural characterization of the lipid A region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The lipid A components of Aeromonas salmonicida subsp. salmonicida from strains A449, 80204-1 and an in vivo rough isolate were isolated by mild acid hydrolysis of the lipopolysaccharide. Structural studies carried out by a combination of fatty acid, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed that the structure of lipid A was conserved among different isolates of A. salmonicida subsp. salmonicida. All analyzed strains contained three major lipid A molecules differing in acylation patterns corresponding to tetra-, penta- and hexaacylated lipid A species and comprising 4'-monophosphorylated beta-2-amino-2-deoxy-d-glucopyranose-(1-->6)-2-amino-2-deoxy-d-glucopyranose disaccharide, where the reducing end 2-amino-2-deoxy-d-glucose was present primarily in the alpha-pyranose form. Electrospray ionization-tandem mass spectrometry fragment pattern analysis, including investigation of the inner-ring fragmentation, allowed the localization of fatty acyl residues on the disaccharide backbone of lipid A. The tetraacylated lipid A structure containing 3-(dodecanoyloxy)tetradecanoic acid at N-2',3-hydroxytetradecanoic acid at N-2 and 3-hydroxytetradecanoic acid at O-3, respectively, was found. The pentaacyl lipid A molecule had a similar fatty acid distribution pattern and, additionally, carried 3-hydroxytetradecanoic acid at O-3'. In the hexaacylated lipid A structure, 3-hydroxytetradecanoic acid at O-3' was esterified with a secondary 9-hexadecenoic acid. Interestingly, lipid A of the in vivo rough isolate contained predominantly tetra- and pentaacylated lipid A species suggesting that the presence of the hexaacyl lipid A was associated with the smooth-form lipopolysaccharide.     

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.     

The outer membrane proteins of the fish pathogens Aeromonas hydrophila, Aeromonas salmonicida and Edwardsiella tarda

Abstract The profiles of the outer membrane proteins contained many major proteins and were markedly different in a number of Aeromonas hydrophila strains isolated from various origins. In contrast, the profiles of outer membrane proteins in Aeromonas salmonicida from different sources were identical and quite different from those of A. hydrophila .
In the strains of Edwardsiella tarda tested, protein components in the outer membrane were quite similar. 2 Major proteins, of 36 and approx. 46 kDa, were found in the outer membrane. Major proteins in A. hydrophila and A. salmonicida were detected in the 32–36 kDa range, as observed in many Enterobacteriaceae. In all strains tested, several 68–90 kDa polypeptides appeared in the outer membrane during iron starvation.     

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.     

Purification and characterization of a new proteolytic enzyme produced by Aeromonas salmonicida

A proteolytic enzyme produced by the fish pathogen Aeromonas salmonicida was isolated and purified. It showed the following characteristics: temperature optimum at 48°C, pH optimum at pH 9 and a molecular weight of 87 500. The enzyme was inhibited by phenylmethanesulphonylfluoride (PMSF) indicating it to be a serine protease.     

O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels

Abstract The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs]) and outer membrane proteins [OMPs] was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB⁺ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB⁻ strains antigenically diverse that either exhibited a heterogenous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB⁺ strains, whereas major OMPs detected in PAB⁻ strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.