The O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila

Abstract We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20 °C but not when they were grown at 37 °C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide ( rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.     

The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34

The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype O:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serum-resistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype O:34 with moderate virulence.     

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34

The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.     

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.     

The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila

A gene encoding a deoxyribonuclease, dnsH, was cloned from Aeromonas hydrophila JMP636. The predicted mature protein was very similar to the previously described extracellular Dns from this organism and an N-terminal region corresponding to a large putative signal sequence was predicted for the JMP636 protein. Inactivation of dnsII demonstrated that the DnsH protein was not present extracellularly in this strain. As DnsH degraded plasmid DNA and was believed to have a periplasmic location, a dnsH mutant was constructed to determine whether electroporation of A. hydrophila with plasmid DNA could be achieved. No transformants were detected. From SDS-PAGE studies, at least two additional DNases remain to be characterised from A. hydrophila JMP636.     

Characterization of Aeromonas hydrophila Strains of Clinical,Animal, and Environmental Origin Expressing the O:34 Antigen

A collection of Aeromonas strains of different origins were characterized for isolates expressing the O:34 somatic antigen. Of over 200 strains tested, approximately 14% belonged to serogroup O:34 with >85% of these strains identified as A. hydrophila regardless of source. A subset of 14 A. hydrophila O:34 strains were further analyzed for a number of structural and pathogenic features. Most O:34 strains expressed similar whole-cell protein profiles with regards to minor bands, but major band differences were noted in outer membrane proteins (OMPs) migrating between the 31K and 58K region. OMP profiles could be subdivided into three distinct patterns. All O:34 strains expressed a heterogeneous O polysaccharide side chain profile in their lipopolysaccharide (LPS), although some variation in the electrophoretic migration of lower and higher molecular weight LPS bands was noted. Polyclonal antisera raised against a 45-K OMP-associated protein of one O:34 strain (AH-195) reacted in immunoblot assays with a major 43 to 46-K OMP in 11 of 14 (79%) O:34 strains tested. Most O:34 strains (69%) were found to be pathogenic in mice with LD-50 values (i.p.) of <1.0 × 10⁷ CFU; pathogenicity appeared to correlate best with elevated protease activity. The collective results suggest significant differences in both structural and pathogenic properties between some members of the O:34 group originating from human and nonhuman (fish, water) sources. Received: 14 December 1995 / Accepted: 22 January 1996     

The regulation of biofilm development by quorum sensing in Aeromonas hydrophila

Aeromonas hydrophila is an opportunistic Gram-negative pathogen that readily attaches to stainless steel to produce a thin biofilm with a complex 3D structure covering 40-50% of the available surface and producing large microcolonies. As A. hydrophila possesses an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system based on the ahyRI locus, the presence of the AhyI protein and C4-HSL within the biofilm phase was first established by Western blot and AHL biosensor analysis respectively. The ability of the A. hydrophila AH-1 N strain to form biofilms in a continuous-flow chamber was compared with isogenic ahyI and ahyR mutants. The ahyI mutant, which cannot produce C4-HSL, failed to form a mature biofilm. In addition, the viable count of biofilm, but not planktonic phase ahyI mutants, was significantly lower that the parent or ahyR mutant. This defect in the differentiation of the ahyI mutant biofilm could be partially restored by the addition of exogenous C4-HSL. A mutation in ahyR increased coverage of the available surface to around 80% with no obvious effect upon biofilm microcolony formation. These data support a role for AHL-dependent quorum sensing in A. hydrophila biofilm development. Exposure of the A. hydrophila AH-1N biofilm to N-(3-oxodecanoyl)homoserine lactone, which inhibits exoprotease production in planktonic cells, however, had no effect on biofilm formation or architecture within the continuous-flow chamber.     

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.     

Structure of the O-polysaccharide of Aeromonas hydrophila O:34; a case of random O-acetylation of 6-deoxy-L-talose

The O-polysaccharide of Aeromonas hydrophila O:34 was obtained by mild-acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy before and after O-deacetylation. The polysaccharide was found to contain D-Man, D-GalNAc and 6-deoxy-L-talose (L-6dTal), and the following structure of the tetrasaccharide repeating unit was established [carbohydrate structure see text] where 6dTal(I) is O-acetylated stoichiometrically at position-2 and 6dTal(II) carries no, one or two O-acetyl groups at any positions.     

Pathogenic Aeromonas hydrophila serogroup O:14 and O:81 strains with an S layer

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10(4.61) to 10(7.11)), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.     

Pathogenic Aeromonas hydrophila Serogroup O:14 and O:81 Strains with an S Layer

Five autoagglutinating Aeromonas hydrophila isolates recovered from eels and humans were assigned to serogroups O:14 and O:81 of the Sakazaki and Shimada (National Institutes of Health) scheme. They had the following properties in common: positive precipitation after boiling, moderate surface hydrophobicity (salt-aggregation-test value around 1.2), pathogenicity for fish and mice (50% lethal dose, 10^4.61 to 10^7.11), lipopolysaccharides that contained O-polysaccharide chains of homogeneous chain length, and an external S layer peripheral to the cell wall observed by electron microscopy. A strong cross-reactivity was detected by immunoblotting between the homogeneous O-polysaccharide fraction of O:14 and O:81 strains but not between them and the lipopolysaccharide of A. hydrophila TF7 (O:11 reference strain). Outer membrane fractions of these strains contained a predominant 53- to 54-kDa protein which was glycine extractable under low-pH (pH 2.8) conditions and was identified as the surface array protein. The S-layer proteins of the O:14 and O:81 A. hydrophila strains seemed to be primarily different from those previously purified from strains A. hydrophila TF7 and Aeromonas salmonicida A450 on the basis of colony hybridizations with both the structural genes vapA and ahsA. This is the first report of the presence of an S layer in mesophilic Aeromonas strains not belonging to serogroup O:11.     

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10⁴–10⁵ cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully detected in infected eels and water samples. We described two subgroups within the serogroup O:19 (Guinée and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group.     

Effect of growth temperature on complement-mediated killing of mesophilic Aeromonas spp. serotype O:34

Abstract Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37°C (serum-sensitive) but not when they were cultivated at 20°C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20°C and rough lipolysaccharide when cultivated at 37°C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.     

Synthesis of Escherichia coli O9a polysaccharide requires the participation of two domains of WbdA, a mannosyltransferase encoded within the wb* gene cluster

WbdA (previously MtfA) is one of the mannosyltransferases encoded within the Escherichia coli O9a wb* gene cluster. It is composed of two domains of similar size, connected by an α-helix chain. Elimination of the C-terminal half by transposon insertion or gene deletion caused synthesis of an altered structural O-polysaccharide consisting only of α-1,2-linked mannose. O9a polysaccharide synthesis was restored by the C-terminal half of WbdA in trans . No membrane incorporation of mannose from GDP mannose was observed in a strain carrying only the gene for truncated WbdA. For mannose incorporation, it was necessary to introduce both wbdB and wbdC genes into the strain. Therefore, it is likely that the N-terminal half of truncated WbdA synthesizes the altered O-polysaccharide together with other mannosyltransferases which participate in the initial reactions of the O9a polysaccharide synthesis. Both N- and C-terminal domains of WbdA are required for the synthesis of the complete E. coli O9a polysaccharide. The chi sequence location between the two domains and homology plot analyses of the wbdA and the WbdA protein suggested that the wbdA gene might have arisen by fusion of two independent genes.     

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus

The effect of temperature on Aeromonas hydrophila infection in goldfish, Carassius auratus , was studied using A. hydrophila strain A-3500. After comparison of four different infection methods, subcutaneous injection was selected. Different test temperatures were also tested and higher mortality was observed at 17 and 25°C during a 15-day period. SDS-PAGE analysis of outer membrane proteins prepared from A. hydrophila cultured at 10, 17, 25 and 32°C in formulated salt water showed different protein profiles. For example, a 40-kDa band was found only at 17 and 25°C. Phagocytic rates of A. hydrophila by goldfish macrophages at 10, 17, 25 and 32°C were 20.46 ± 2.07, 16.15 ± 1.39, 15.94 ± 1.85 and 22.22 ± 2.49%, respectively. The results indicated that temperature affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish macrophages, resulting in varying fish mortality when infected at different temperatures.     

Molecular detection and cloning of thermostable hemolysin gene from Aeromonas hydrophila

Aeromonas hydrophila is a major bacterial pathogen associated with hemorrhagic septicemia in aquatic and terrestrial animals including humans. There is an urgent need to develop molecular and immunological assays for rapid, specific and sensitive diagnosis. A new set of primers has been designed for detection of thermostable hemolysin (TH) gene (645 bp) from A. hydrophila, and sensitivity limit for detection of TH gene was 5 pg. The TH gene was cloned, sequenced and analyzed. The G+C content was 68.06%; and phylogeny was constructed using TH protein sequences which had significant homology with those for thermostable and other hemolysins present in several bacterial pathogens. In addition, we have predicted the four and eight T-cell epitopes for MHC class I and II alleles, respectively. These results provide new insight for TH protein containing antigenic epitopes that can be used in immunoassays and also designing of thermostable vaccines.     

Effect of temperature and arsenic on Aeromonas hydrophila growth,a modelling approach

Aeromonas hydrophila is frequently reported from arsenic affected areas. Present study was aimed to determine the effect of arsenic and temperature on growth of A. hydrophila. The bacteria were isolated from naturally infected fish from a water body in Birbhum, West-Bengal, India, which is reported to be an arsenic-free area. Arsenic concentration in natural aquatic reservoirs (e.g., pond, lake or river) varies from 0–6 mg/L. No significant change in bacterial growth was observed within this range of arsenic exposure. However, variation in temperature impacted the growth of A. hydrophila. A single dimension model was constructed using simple logistic equation. Rate parameters of the model were derived from the experimental observations. Comparison of model results and laboratory observations gives a good conformity regarding the effect of variation of arsenic concentration and temperature change on growth of this bacterium. From the analysis of this model we further get the idea that the maximum growth of A. hydrophila is supposed to be at 31.4°C in absence of arsenic, whereas at 477 mg/L arsenic concentration, the growth of the bacteria totally stops at 30°C.     

Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila.

A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product.     

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.