Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Structural insights into acyl-ACP selective recognition by the Aeromonas hydrophila AHL synthase AhyI.

BACKGROUND: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase. RESULTS: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay. CONCLUSIONS: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Extracellular electron transfer via multiple electron shuttles in waterborne Aeromonas hydrophila for bioreduction of pollutants.

Members of the genus Aeromonas prevail in aquatic habitats and have a great potential in biological wastewater treatment because of their unique extracellular electron transfer (EET) capabilities. However, the mediated EET mechanisms of Aeromonas have not been fully understood yet, hindering their applications in biological wastewater treatment processes. In this study, the electron shuttles in Aeromonas hydrophila, a model and widespread strain in aquatic environments and wastewater treatment plants, were explored. A. hydrophila was found to produce both flavins and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as electron shuttles and utilize them to accelerate its EET for the bioreduction of various pollutants. The Mtr-like respiratory pathway was essential for the reduction of flavins, but not involved in the ACNQ reduction. The electron shuttle activity of ACNQ for pollutant bioreduction involved the redox reactions that occurred inside the cell. These findings deepen our understanding about the underlying EET mechanisms in dissimilatory metal reducing bacteria and provide new insights into the roles of the genus Aeromonas in biological wastewater treatment.

Acetylation of lysine 7 of AhyI affects the biological function in Aeromonas hydrophila.

Acyl-homoserine-lactone synthase (AhyI) of Aeromonas hydrophila can produce quorum sensing (QS) auto-inducer 1 (AI-1) type signal molecule, which plays important roles in various biological phenomenons such as biofilm formation, hemolysin production and motility. Previous research revealed that the AhyI of A. hydrophila has acetylation modification on lysine 7 site, but its intrinsic biological function is still largely unknown. To study the effect of AhyI protein and its acetylation modification on the physiological traits of A. hydrophila, the site-directed mutagenesis strains including ΔahyI::ahyI-K7Q and ΔahyI::ahyI-K7R were made in this study. The mutation at K7 site of lysine acetylation in AhyI protein decreased the protease production, but the lysine acetylations do not affect the biofilm formation and hemolysin production. To further study the effect of lysine acetylation on AI-1 signal molecule production, the acyl-homoserine lactones (AHLs) extraction and bioluminescence quantification were performed. Compared with the rescue strain, the acetylation on K7 of AhyI resulted in a decreased level of AHLs and bioluminescence production. It indicated that the lysine acetylation modification on the AhyI protein can regulate the production of signalling molecules. Overall, the obtained data in this study provide a theoretical basis for further understanding the role of lysine acetylation of AhyI protein and lay a foundation to systematically study the regulatory mechanism of QS.

Comparative proteomic analysis of sensitive and multi-drug resistant Aeromonas hydrophila isolated from diseased fish.

Bacterial hemorrhagic septicemia caused by multi-drug resistant (MDR) Aeromonas hydrophila has exponentially increased in the past decade, and reached an alarming rate making it a major concern in the aquaculture industry in China. The aim of this study was to investigate the difference in the regulation of proteins expression in multi-drug resistance and susceptible A. hydrophila strains isolated from diseased fish using two-dimensional electrophoresis (2-DE) combined with mass spectrometry. 28 isolates of A. hydrophila were successfully identified by biochemical tests. Antibiotic susceptibility test results showed that all the isolates have different drug resistant patterns. A total of 61 and 17 differently expressed proteins were identified in MDR and susceptible A. hydrophila, respectively, evidencing that biological processes related to carbon metabolism, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, cationic antimicrobial peptide (CAMP) resistance and propanoate metabolism were down-regulated in MDR strain, while proteins involved in biosynthesis of antibiotics, glycolysis/gluconeogenesis were highly expressed in the sensitive strain. The analysis of differentially expressed proteins from multi-drug resistance and susceptible strains suggests that a number of proteins are involved in several metabolic metabolism pathways plays an important role in A. hydrophila drug resistance. Our findings provide new insights about mechanisms involved in drug resistance and propose possible novel targets for developing alternative antibacterial drugs.

Crystal Structure of Aeromonas hydrophila Cytoplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine (MTA/SAH) nucleosidase (MTAN) is an important enzyme in a number of critical biological processes. Mammals do not express MtaN, making this enzyme an attractive antibacterial drug target. In pathogen Aeromonas hydrophila, two MtnN subfamily genes (MtaN-1 and MtaN-2) play important roles in the periplasm and cytosol, respectively. We previously reported structural and functional analyses of MtaN-1, but little is known regarding MtaN-2 due to the lack of a crystal structure. Here, we determined the crystal structure of cytosolic A. hydrophila MtaN-2 in complex with adenine (ADE), which is a cleavage product of adenosine. AhMtaN-1 and AhMtaN-2 exhibit a high degree of similarity in the α-ß-α sandwich fold of the core structural motif. However, there is a structural difference in the nonconserved extended loop between ß7 and α3 that is associated with the channel depth of the substrate-binding pocket and dimerization. The ADE molecules in the substrate-binding pockets of AhMtaN-1 and AhMtaN-2 are stabilized with π-π stacking by Trp199 and Phe152, respectively, and the hydrophobic residues surrounding the ribose-binding sites differ. A structural comparison of AhMtaN-2 with other MtaN proteins showed that MtnN subfamily proteins exhibit a unique substrate-binding surface and dimerization interface.

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking.

The molecular recognition and interaction of CphA from Aeromonas hydrophila with imipenem (Imip) and biapenem (Biap) were studied by means of the combined use of fluorescence spectra and molecular docking. The results showed that both the fluorescence quenching of CphA by Imip and Biap were caused through the combined dynamic and static quenching, and the latter was dominating in the process; the microenvironment and conformational of CphA were altered upon the addition of Imip and Biap from synchronous and three-dimensional fluorescence. The binding of CphA with Imip or Biap caused a conformational change in the loop of CphA, and through the conformational change, the loop opened the binding pocket of CphA to allow for an induced fit of the newly introduced ligand. In the binding of CphA with Imip, the whole molecule entered into the active pocket of CphA. The binding was driven by enthalpy change, and the binding force between them was mainly hydrogen bonding and Van der Waals force; whereas in the binding of CphA with Biap, only the beta-lactam ring of Biap entered into the binding pocket of CphA while the side chain was located outside the active pocket. The binding was driven by the enthalpy change and entropy change together, and the binding force between them was mainly electrostatic interaction. This study provided an insight into the recognition and binding of CphA with antibiotics, which may be helpful for designing new substrate for beta-lactamase and developing new antibiotics resistant to superbugs.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→ O-PS2 →2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Direct Sensing of Single Native RNA with a Single-Biomolecule Interface of Aerolysin Nanopore.

RNA sensing is of vital significance to advance our comprehension of gene expression and to further benefit medical diagnostics. Taking advantage of the excellent sensing capability of the aerolysin nanopore as a single-biomolecule interface, we for the first time achieved the direct characterization of single native RNA of Poly(A)4 and Poly(U)4. Poly(A)4 induces ∼10% larger blockade current amplitude than Poly(U)4. The statistical duration of Poly(A)4 is 18.83 ± 1.08 ms, which is 100 times longer than that of Poly(U)4. Our results demonstrated that the capture of RNA homopolymers is restricted by the biased diffusion. The translocation of RNA needs to overcome a lower free-energy barrier than that of DNA. Moreover, the strong RNA-aerolysin interaction is attributed to the hydroxyl in pentose, which prolongs the translocation time. This study opens an avenue for aerolysin nanopores to directly achieve RNA sensing, including discrimination of RNA epigenetic modification and selective detection of miRNA.

Structural investigation by tandem mass spectrometry analysis of a heterogeneous mixture of Lipid An isolated from the lipopolysaccharide of Aeromonas hydrophila SJ-55Ra.

RATIONALE: We report herein the electrospray ionization mass spectrometry (ESI-MS) negative ion mode and low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) analysis of a mixture of lipid An isolated from the lipopolysaccharide (LPS) of a rough-resistant wild strain of the Gram-negative bacteria Aeromonas hydrophila grown in the presence of phages (SJ-55Ra). This investigation indicates that the presence of a mixture of lipid A acylated disaccharides, whose molecular structures were not relatively conserved, resulted from the incomplete LPS biosynthesis caused by the phage treatment. METHODS: The heterogeneous lipid An mixture from the LPS-SJ55Ra was obtained following growth of the Gram-negative bacteria Aeromonas hydrophila (SJ-55R) in the presence of phages and isolation by the aqueous phenol method. Following hydrolysis and purification of the lipopolysaccharide, ESI-MS and low-energy CID-MS/MS analyses were performed on a triple-quadrupole (QqQ) and a Fourier transform ion cyclotron resonance (FTICR) instrument. RESULTS: ESI-MS analysis suggested that this lipid An mixture contained eight molecular disaccharide anions and three monosaccharide anions. This series of lipid An was asymmetrically substituted with ((R)-14:0(3-OH)) fatty acids located at O-3 and N-2 and with branched fatty acids: (Cl4:0(3-(R)-O-C14:0)) and (C12:0(3-(R)-O-(14:0)) at the O-3' and N-2' positions. CONCLUSIONS: Tandem mass spectrometric analyses allowed the exact determination of the fatty acid acylation locations on the D-GlcpN disaccharide. The MS/MS results established that it was possible to selectively cleave C-O, C-N, and C-C bonds, together with glycosidic C-O and cross-ring cleavages, affording excellent structural analysis of lipid A biomolecules.

Translocation of Precision Polymers through Biological Nanopores.

Nanopore analysis, which is, currently, chiefly used for DNA sequencing, is also an appealing technique for characterizing abiotic polymers. As a first step toward this goal, nanopore detection of non-natural monodispersed poly(phosphodiester)s as candidate backbone structures is reported herein. Two model homopolymers containing phosphopropyl repeat units (i.e., 56 or 104 r.u.) and a short thymidine nucleotide sequence are analyzed in the present work. They are tested in two different biological nanopores, α-hemolysin from Staphylococcus aureus, and aerolysin from Aeromonas hydrophila. These recordings are performed in aqueous medium at different KCl concentrations and various driving voltages. The data show a complex interaction with evidence for voltage dependence and threading, and underline the influence of the molecular structure and orientation of the precision poly(phosphodiester)s on the observed residual current signal as well as on the translocation dynamics. In particular, they suggest a dominant entropic contribution due to the high flexibility of the phosphodiester homopolymer.

Cytotoxic effects of Aeromonas hydrophila culture supernatant on peripheral blood leukocytes of Nile tilapia (Oreochromis niloticus): Possible presence of a secreted cytotoxic lectin.

Number of exotoxins like haemolysin, leukocidin, aerolysin etc. were reported from Aeromonas hydrophila. In this study, we report the haemolytic and cytotoxic effect of A. hydrophila culture supernatant (CS) that is specifically inhibited by lactose and also by serum and mucus of Nile tilapia (Oreochromis niloticus). Hence, we assume the presence of a secreted lectin in the CS. CS is toxic to peripheral blood leukocytes (PBL) of O. niloticus as revealed by MTT assay and by flow cytometry. DNA laddering assay indicates that CS causes necrosis to PBL. As a result of necrosis, CS treated PBL showed increased production of reactive oxygen species as indicated by nitroblue tetrazolium and 2',7' -dichlorofluorescin diacetate assays. CS treated PBL showed reduced mRNA expression of TNF-α and IFN-γ genes. When CS was subjected to polyacrylamide gel electrophoresis, it showed a single band corresponding to the molecular weight of 45 kDa. However, upon concentrating the CS by ultrafiltration, many bands were visualized. Further studies at molecular level are required to unravel this macromolecular-leukocyte interaction which would ultimately benefit the aquaculture industry.

Ecofriendly biosorption of dyes and metals by bacterial biomass of Aeromonas hydrophila RC1.

The ability of dried bacterial biomass in azo dye and heavy metal removal from aqueous solution was explored. Biosorption of three textile dyes, Eriochrome black T (EBT), Acid Red 26 (AR) and Trypan blue (TB) and heavy metals (Pb and Cr) by dried biomass of Aeromonas hydrophila RC1, was investigated in a batch system under various parameters such as dye concentration, contact time, concentration of biomass, pH, and temperature. The experimental results showed that the extent of biosorption for dyes increased with increase in initial concentration of dyes, biomass concentration, contact time, temperature and decreased with increase in pH. The experimental isotherms data were analyzed using Langmuir and Freundlich isotherm equations. The Langmuir model yielded good fit to the experimental data (R² approximately 0.794, 0.844 and 0.969 for the dyes, EBT, AR and TB, respectively) with maximum monolayer adsorption capacity of 58.8 mg g⁻¹ for AR. Similarly results were obtained for heavy metals and the data fit in Langmuir model (R² value of 0.849 and 0.787) with q(m) value of 40 mg g⁻¹ for Pb. The results fit in pseudo first order kinetics with removal upto 96.67 % for Pb. Involvement of the surface characteristics of the biomass in biosorption was studied using scanning electron micrographs, FTIR, EDX and XRD analysis. Thus, use ofA. hydrophila RC1 biomass can be extensively employed in water treatment plants in order to get desired water quality in the most economical way.

The structure of the proteinaceous inhibitor PliI from Aeromonas hydrophila in complex with its target lysozyme.

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Šresolution crystal structure of the periplasmic i-type lysozyme inhibitor from Aeromonas hydrophila (PliI-Ah) in complex with the i-type lysozyme from Meretrix lusoria is reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key-lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme-inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key-lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.

Molecular and chemical analysis of the lipopolysaccharide from Aeromonas hydrophila strain AH-1 (Serotype O11).

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.

[Construction and application of bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer].

As a key component of life science, bioinformatics has been widely applied in genomics, transcriptomics, and proteomics. However, the requirement of high-performance computers rather than common personal computers for constructing a bioinformatics platform significantly limited the application of bioinformatics in aquatic science. In this study, we constructed a bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer. The platform consisted of three functional modules, including genomic and transcriptomic sequencing data analysis, protein structure prediction, and molecular dynamics simulations. To validate the practicability of the platform, we performed bioinformatic analysis on aquatic pathogenic organisms. For example, genes of Flavobacterium johnsoniae M168 were identified and annotated via Blast searches, GO and InterPro annotations. Protein structural models for five small segments of grass carp reovirus HZ-08 were constructed by homology modeling. Molecular dynamics simulations were performed on out membrane protein A of Aeromonas hydrophila, and the changes of system temperature, total energy, root mean square deviation and conformation of the loops during equilibration were also observed. These results showed that the bioinformatic analysis platform for aquatic pathogen has been successfully built on the MilkyWay-2 supercomputer. This study will provide insights into the construction of bioinformatic analysis platform for other subjects.

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a gram-negative bacterium.

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.

Fermentation preparation of recombinant Vibrio anguillarum vaccine with heterogeneous antigen display.

In the design of recombinant bacterial vector vaccine, heterogeneous antigen is displayed on the outer membrane of the vector strain to evoke polyvalent immunological protection. Thus, the expression of heterogeneous antigen in cells and its display on the outer membrane are of great concern for vaccine preparation. In our previous work, a multivalent bacterial vector vaccine MVAV6203A-1 was constructed by displaying the protective antigen GAPDH from Aeromonas hydrophila on the surface of an attenuated Vibrio anguillarum MVAV6203. In this work, a new fermentation medium was designed by a four-step method to improve the cell growth and antigen display of V. anguillarum MVAV6203A-1. First, suitable carbon and nitrogen sources were selected by a component swapping method. Second, the initial concentrations of carbon and nitrogen sources were determined by orthogonal design. Then three main factors to significantly affect cell growth and antigen expression were screened by a Plackett-Burman design. Finally, the three main factors were meticulously optimized by response surface methodology. Based on this medium, a fed-batch fermentation process was established in a 5-L bioreactor, and the dry cell weight, the antigen expression in cells, and its display on outer membrane reached 5.98 g/L, 2.82 mg/g DCW, and 0.119 mg/g DCW, respectively.

Immunochemical studies and gene cluster relationships of closely related O-antigens of Aeromonas hydrophila Pt679, Aeromonas popoffii A4, and Aeromonas sobria K928 strains classified into the PGO1 serogroup dominant in Polish aquaculture of carp and rainbow trout.

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined: [Formula: see text] The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes. For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.

ExeA binds to peptidoglycan and forms a multimer for assembly of the type II secretion apparatus in Aeromonas hydrophila.

Aeromonas hydrophila uses the type II secretion system (T2SS) to transport protein toxins across the outer membrane. The inner membrane complex ExeAB is required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan-binding domain (Pfam number PF01471) conserved in many peptidoglycan-related proteins is present in the periplasmic region of ExeA (P-ExeA). In this study, co-sedimentation analysis revealed that P-ExeA was able to bind to highly pure peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in native PAGE, gel filtration and cross-linking experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at 30 degrees C and above. These results provide evidence that the putative peptidoglycan-binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions facilitate the multimerization of ExeA, favouring a model in which the protein forms a multimeric structure on the peptidoglycan during the ExeAB-dependent assembly of the secretin multimer in the outer membrane.