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PubMed2004

Structural studies on the R-type lipopolysaccharide of Aeromonas hydrophila.

A rough strain of Aeromonas hydrophila, AH-901, has an R-type lipopolysaccharide with the complete core. The following core structure was established by chemical degradations followed by sugar and methylation analyses along with ESIMS and NMR spectroscopy: [formula: see text] where D-alpha-D-Hep and l-alpha-D-Hep stand for D-glycero- and l-glycero-alpha-D-manno-heptose, respectively; Kdo stands for 3-deoxy-D-manno-oct-2-ulosonic acid; all monosaccharides are in the pyranose form; the degree of substitution with beta-D-Gal is approximately 50%. Lipid A of the lipopolysaccharide has a 1,4(')-bisphosphorylated beta-D-GlcN-(1-->6)-alpha-D-GlcN disaccharide backbone with both phosphate groups substituted with 4-amino-4-deoxyarabinose residues.

Aeromonas hydrophila↗
PubMed2005

Structural and serological characterization of the O-chain polysaccharide of Aeromonas salmonicida strains A449, 80204 and 80204-1.

The O-chain polysaccharide (O-PS) of Aeromonas salmonicida was studied by a combination of compositional, methylation, CE-ESMS and one- and two-dimensional NMR analyses. It was found to be a branched polymer of trisaccharide-repeating units composed of L-rhamnose (Rha), D-glucose (Glc), 2-acetamido-2-deoxy-D-mannose (ManNAc) and O-acetyl group (OAc) and having the following structure: CE-ESMS analysis of A. salmonicida cells from strains A449, 80204 and 80204-1 grown under different conditions confirmed that the O-PS structure was conserved. ELISA-based serological study with native LPS-specific antisera performed on the native O-PS and its O-deacetylated and periodate-oxidized derivatives confirmed the importance of the O-PS backbone structure as an immunodominant determinant.

Aeromonas salmonicida↗
PubMed2005

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.

Aeromonas salmonicida↗
PubMed2006

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.

Acylation↗
PubMed2026

Liver transcriptome analysis revealed multiple immune processes and lipid metabolism pathways involved in the defense response of the turbot (Scophthalmus maximus) against Aeromonas salmonicida.

Aeromonas salmonicida is a significant pathogen causing notable economic losses in Scophthalmus maximus aquaculture. This study utilized Illumina sequencing technology to examine the transcriptional response characteristics of S. maximus liver at 24 h following A. salmonicida infection. A total of 2363 differentially expressed genes (DEGs) were identified when compared to the negative control group. The immunity-related Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, as well as metabolism-related PPAR signaling pathway and insulin signaling pathway, were notably enriched. Significant differences exist in the expression of key genes within the PPAR pathway, particularly cd36, acsl4a, pparαa, and plin2, all of which mediate the interaction between lipid metabolism and the immune response. These results offer valuable insights into the immunometabolic regulatory mechanism of S. maximus response to A. salmonicida infection.

Animals↗
PubMed2005

Factors affecting biosurfactant production by oil degrading Aeromonas spp. isolated from a tropical environment.

An Aeromonas spp. was isolated from tropical estuarine water. The organism grew on crude oil and produced biosurfactant that could emulsify hydrocarbons. The peak growth and biosurfactant production was on the 8th day. The organism grew on a range of hydrocarbons that include crude oil and hexadecane while no growth was recorded on some hydrocarbons that include benzene. The biosurfactant produced by the organism emulsified a range of hydrocarbons with diesel (E24=65) as the best substrate and hexane (E24=22) as the poorest. After purification, the biosurfactant was found to contain about 38% carbohydrate and an unidentified lipid. No protein was present in the purified biosurfactant. Production of biosurfactant was highest in medium with glucose and lowest in the medium with diesel+acetate. Soybean was the best nitrogen source for biosurfactant production. The activity of the biosurfactant was enhanced optimally at NaCl concentration of 5%, pH of 8.0 and temperature of 40 degrees C. The biosurfactant retained 77% of its original activity after 120 min of exposure to heat at a temperature of 100 degrees C. Biosurfactant may be produced with this organism using non-hydrocarbon substrates such as glucose and soybean that are readily available and would not require extensive purification for use in food and pharmaceutical industries.

Aeromonas↗
PubMed2006

Immune modulation by probiotic strains: quantification of phagocytosis of Aeromonas salmonicida by leukocytes isolated from gut of rainbow trout (Oncorhynchus mykiss) using a radiolabelling assay.

A method is described for the rapid and sensitive assay of phagocytosis that utilizes radioactively labelled bacteria. With this method, we observed that phagocytosis of heat-inactivated Aeromonas salmonicida by leukocytes isolated from gut of rainbow trout fed with different viable probiotics (Lactococcus lactis subsp. lactis, Lactobacillus sakei, and Leuconostoc mesenteroides) was significantly higher (P<0.05) after 2 weeks of probiotic-feeding than the control group. However, only phagocytosis of live A. salmonicida by the leukocytes isolated from gut of rainbow trout fed with L. lactis subsp. lactis was significantly higher (P<0.05) than the control group.

Aeromonas salmonicida↗
PubMed2005

Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology.

The response of Atlantic salmon, Salmo salar, to infection by the bacterial pathogen Aeromonas salmonicida (the causative agent of furunculosis), was investigated using a cohabitation model and a custom Atlantic salmon cDNA microarray consisting of over 4000 different amplicons. Pooled samples of each of three immune-relevant tissues (spleen, head kidney and liver) were obtained from fish exposed to infected salmon for 13 days. Reverse transcription-PCR assays were used to verify the differential expression of 12 candidate genes uncovered by microarray analysis. Among the differentially expressed genes were several previously revealed by suppression subtractive hybridization and EST surveys and that are recognized to encode humoral components of the innate immune system. Other genes identified in this study were not previously associated with infection. In addition, a number of genes with no known homologs were uncovered. Determination of their specific roles during infection may lead to a better understanding of innate immunity.

Aeromonas salmonicida↗
PubMed2005

Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish.

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 degrees C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.

Aeromonas salmonicida↗
PubMed2006

Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Aeromonas salmonicida and Vibrio anguillarum.

The immune system of Atlantic cod (Gadus morhua L.) differ from other bony fish species in that no or only very low increases in antibody levels are detected post-immunization with Vibrio salmonicida or V. anguillarum. Here, we report the results from the first study on comparison of antibody responses in cod to Aeromonas salmonicida and V. anguillarum. A. salmonicida appear to induce a stronger antibody response in cod compared to V. anguillarum, and more individuals immunized with A. salmonicida revealed a response compared to those immunized with V. anguillarum. The antibody responses to both bacterial species were mainly toward LPS, and the results indicate that cod antibodies are able to differentiate between LPS antigens from typical and atypical A. salmonicida strains.

Aeromonas salmonicida↗
PubMed2006

Characterization of twin toll-like receptors from rainbow trout (Oncorhynchus mykiss): evolutionary relationship and induced expression by Aeromonas salmonicida salmonicida.

Structure and function of factors contributing to the innate immune system of lower vertebrates, including fish are only sparsely characterized. We retrieved with RT-PCR cDNA copies of two closely related Toll-like receptors (TLR) from liver RNA of the rainbow trout (Oncorhynchus mykiss). The cDNA sequences are homologous to 95.6%. The phylogenetic analysis of their deduced amino acid sequences places these twin factors closely to other known TLRs from fish. The twin factors are equally expressed in all tissues analysed, most abundantly in spleen and head kidney and lowest in adipose tissue. Formalin-inactivated Aeromonas salmonicida pathogens induce their expression up to eight-fold in vitro in peripheral blood lymphocytes and in tissues from spleen and head kidney. Our sequence information will be useful to establish expression constructs for these factors necessary to analyse the pathogen specific signal transduction activating the innate immune defence in fish.

Aeromonas salmonicida↗
PubMed2026

Genome-wide characterization of NOD-like receptor genes links NLR repertoire evolution to spleen immune responses after Aeromonas hydrophila challenge in the Chinese spiny frog (Quasipaa spinosa).

NOD-like receptors (NLRs) are cytosolic pattern-recognition receptors that detect pathogen-associated and damage-associated molecular patterns and mediate innate immune signaling in vertebrates. However, the genomic repertoire, evolutionary diversification, and infection-associated expression of NLR genes remain poorly defined in non-model amphibians. In this study, 66 NLR genes were identified from the Chinese spiny frog (Quasipaa spinosa) genome and designated as QsNLR1-QsNLR66. These genes were unevenly distributed across chromosomes and were classified into three phylogenetic groups, with most members exhibiting conserved motif architectures. Gene duplication analysis indicated that dispersed duplication was the main contributor to QsNLR expansion. Synteny analysis detected five conserved orthologous gene pairs between Q. spinosa and Pelophylax nigromaculatus, suggesting partial conservation of NLR genomic organization between the two amphibians. Ka/Ks analysis showed that several duplicated gene pairs, including NLRC3-like/QsNLR36 and NLRC3-like/QsNLR50, exhibited Ka/Ks ratios greater than one, suggesting potential sequence divergence after duplication. Spleen RNA sequencing (RNA-seq) after Aeromonas hydrophila challenge revealed enrichment of immune-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Weighted gene co-expression network analysis linked several QsNLRs to infection-associated modules, among which QsNLR57 was co-expressed with CYBB, ADAM17, SPI1, and HK2. RT-qPCR using time-matched phosphate-buffered saline (PBS) controls showed distinct temporal patterns, with stronger induction of QsNLR29, QsNLR57, and QsNLR66 and weaker or delayed responses of QsNLR50 and QsNLR56. These results characterize the NLR repertoire of Q. spinosa and identify infection-associated QsNLR candidates for future studies of antibacterial immunity in amphibians.

Animals↗
PubMed2004

The Aeromonas hydrophila LuxR homologue AhyR regulates the N-acyl homoserine lactone synthase, AhyI positively and negatively in a growth phase-dependent manner.

Aeromonas hydrophila is a pathogen of fish, amphibians and humans which produces N-acylhomoserine lactone quorum sensing signal molecules and possesses homologues of the Vibrio fischeri luxI and luxR quorum sensing genes termed ahyI and ahyR, respectively. The ahyI and ahyR genes of A. hydrophila comprise a divergon with a 62 bp intergenic region and control biofilm maturation and extracellular protease production. Stationary phase culture supernatants from an ahyR but not an ahyI mutant contain N-butanoylhomoserine lactone (C4-HSL) which is shown to be required for maximal ahyI expression. To determine whether AhyR regulates ahyI, the expression of AhyI was followed throughout growth by Western blot analysis. This revealed that AhyI can be detected in the exponential phase but appears to be degraded in stationary phase in the parent A. hydrophila strain. In an ahyR mutant however, the AhyI protein is only produced in stationary phase but production is sustained suggesting that AhyR controls the timing of AhyI production and turnover. By using RT-PCR, we mapped the transcriptional start site of ahyI which revealed that the 12 bp symmetrical lux-box like sequence present in the 62 bp ahyRI intergenic region overlaps with the -10 region of the ahyI promoter. To determine whether AhyR could bind to the ahyRI intergenic region, the ahyR gene was expressed and purified as a maltose binding protein (MalE) fusion. Electrophoretic mobility shift assays demonstrated that MalE-AhyR specifically bound to this sequence in both the presence and absence of N-butanoylhomoserine lactone (C4-HSL). Taken together, these data suggest that AhyR acts as both a negative and a positive regulator of ahyI and hence C4-HSL production in a growth phase dependent manner.

4-Butyrolactone↗
PubMed2005

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida.

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.

Aeromonas salmonicida↗
PubMed2005

A pore-forming toxin produced by Aeromonas sobria activates cAMP-dependent Cl- secretory pathways to cause diarrhea.

Aeromonas sobria hemolysin (ASH) is one of the major virulence factors produced by A. sobria, a human pathogen that causes diarrhea. We investigated the effects of ASH on Cl(-) transport in human colonic epithelial cells. ASH increased short-circuit currents (Isc) and (125)I efflux from Caco-2 cells, indicating ASH activate Cl(-) secretion. Additions of inhibitors of cyclic AMP dependent Cl(-) channels, glybenclamide and NPPB suppressed the Isc and (125)I efflux increases induced by ASH. And ASH increased the intracellular cyclic AMP concentration. Moreover, ASH stimulated fluid accumulation in the iliac loop test, and glybenclamide and NPPB suppressed this fluid accumulation. Thus, cAMP-dependent Cl(-) secretory pathway could be related with diarrhea induced by A. sobria.

Aeromonas↗
PubMed2005

Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes.

Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.

Aeromonas hydrophila↗
PubMed2005

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp.

Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.

3-Hydroxybutyric Acid↗
PubMed2005

Effect of over-expression of phasin gene from Aeromonas hydrophila on biosynthesis of copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate.

The gene phaPAh, encoding the protein phasin that is associated with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) granule of Aeromonas hydrophila 4AK4, was cloned and characterized. Recombinant strains harboring additional copies of the phasin gene (phaPAh) and the polyhydroxyalkanoate (PHA) synthase gene (phaCAh) accumulated PHBHHx copolyesters consisting of 21 mol% 3-hydroxyhexanoate (3HHx) as compared to 14 mol% 3HHx produced by wild type strain. The molecular weight of PHBHHx produced by the above recombinants was lower than that obtained from the wild type strain grown under similar conditions. Over-expression of phaPAh led to the production of more PHA granules but with reduced sizes. SDS-PAGE showed that PhaPAh was the predominant protein present in the PHBHHx granules. The RT-PCR results suggested that phasin PhaPAh, regulated phaCAh gene at the transcription level. Gene PhaPWe from Wautersia eutropha (formerly Ralstonia eutropha; encoding a 20 kDa protein with low amino acid homology to the A. hydrophila 13 kDa protein) cloned into A. hydrophila 4AK4 exhibited similar effects on PHBHHx production and PHBHHx composition. These data suggest that the phasins could represent a protein family possessing similar functions but different structures.

3-Hydroxybutyric Acid↗