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.

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.

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.

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.

Insights into the Inhibition of Aeromonas hydrophila d-Alanine-d-Alanine Ligase by Integration of Kinetics and Structural Analysis.

Aeromonas hydrophila, a pathogenic bacterium, is harmful to humans, domestic animals, and fishes and, moreover, of public health concern due to the emergence of multiple drug-resistant strains. The cell wall has been discovered as a novel and efficient drug target against bacteria, and d-alanine-d-alanine ligase (Ddl) is considered as an essential enzyme in bacterial cell wall biosynthesis. Herein, we studied the A. hydrophila HBNUAh01 Ddl (AhDdl) enzyme activity and kinetics and determined the crystal structure of AhDdl/d-Ala complex at 2.7 Å resolution. An enzymatic assay showed that AhDdl exhibited higher affinity to ATP (Km: 54.1 ± 9.1 µM) compared to d-alanine (Km: 1.01 ± 0.19 mM). The kinetic studies indicated a competitive inhibition of AhDdl by d-cycloserine (DCS), with an inhibition constant (Ki) of 120 µM and the 50% inhibitory concentrations (IC50) value of 0.5 mM. Meanwhile, structural analysis indicated that the AhDdl/d-Ala complex structure adopted a semi-closed conformation form, and the active site was extremely conserved. Noteworthy is that the substrate d-Ala occupied the second d-Ala position, not the first d-Ala position. These results provided more insights for understanding the details of the catalytic mechanism and resources for the development of novel drugs against the diseases caused by A. hydrophila.

Computer aided novel antigenic epitopes selection from the outer membrane protein sequences of Aeromonas hydrophila and its analyses.

OBJECTIVES: Gram-negative bacteria are among the causative microorganisms for zoonotic diseases in humans and teleosts. Outer membrane proteins (Omps) of Aeromonas hydrophila, a gram-negative bacterium, are critical for the subcellular integration to eukaryotic cell that can modulate the functions of macrophages. Hence Omps are recognized as immune markers for the vaccine development. METHODS: In the present study, a 3-D model of Omps was identified using in silico technique and recognized through the Swiss model web-server and confirmed with Procheck and ProSA server.. The B-cell binding sites of the protein were selected from sequence alignment.. Further, the identification of B-cell epitope was carried out using modules of BCpred server (i.e., BCPred and Amino Acid Pairs). The identified antigenic amino acid sequences for B-cells were used to determine the T-cell epitope (both MHC I & II allelic binding sequences) using ProPred 1 and ProPred servers. RESULTS: The epitopic regions (9 mer: LAGKTTNES and GFDGSQYGK) in the Omps that are bound together with the MHC molecules (MHC-I & II), and have maximum possible numbers of MHC alleles are recognized. It was observed that Omps of A. hydrophila are conserved across the serotypes and are immunogenic. These epitopes can stimulate significant immune responses and can be advantageous while preparing peptide-based vaccines against A. hydrophila infections. Thus, suggesting the use of Omps in the development of vaccines and immunotherapeutics against the bacterial diseases in humans and teleosts.

Identification of a Novel N-Acyl Homoserine Lactone Synthase, AhyI, in Aeromonas hydrophila and Structural Basis for Its Substrate Specificity.

In the Gram-negative bacterium Aeromonas hydrophila, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the ahyI gene from the isolate A. hydrophila HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces N-butanoyl-l-homoserine lactone (C4-HSL) and N-hexanoyl-l-homoserine lactone (C6-HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C4-HSL using S-adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the A. hydrophila HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a ß bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C4-ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.

Use of bacterial cellulose film modified by polypyrrole/TiO2-Ag nanocomposite for detecting and measuring the growth of pathogenic bacteria.

The aim of this study was to use of bacterial cellulose/polypyrrole/TiO2-Ag (BC/PPy/TiO2-Ag) nanocomposite film to detect and measure the growth of 5 pathogenic bacteria. For this purpose, at first, 13 BC/PPy/TiO2-Ag films were fabricated, then bacterial suspensions were prepared according to McFarland standard. The results showed that by increasing the bacterial concentration, the electrical resistance of sensors was decreased and there was a relation between bacterial concentration and bacterial type with electrical resistance change of sensors. The obtained data showed that the sensitivity of the sensors was increased with increasing the concentration of polypyrrole and TiO2-Ag. FT-IR and SEM tests were performed to investigate the interaction between nanoparticles and determine the size of nanoparticles. The BC/PPy/TiO2-Ag biosensors are portable and the response time of these sensors is very short for target analysis. Therefore, these sensors have the potential to improve biological safety as diagnostic tools.

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.

Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila.

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent responsible for disease outbreaks in farmed carp species and channel catfish (Ictalurus punctatus) in China and the Southeastern United States, respectively. The possible route of infection has previously been unknown; however, virulence is believed to be multifactorial, involving the production/secretion of several virulence factors, including a high molecular weight group 4 capsular polysaccharide. Here we present chemical structural evidence of a novel capsule- and LPS-associated O-antigen found present in vAh isolated during these disease outbreaks. In this study, the chemical structure of the vAh O-antigen was determined by chemical analysis, Smith degradation, mass spectrometry, and 2D proton and carbon nuclear magnetic resonance (NMR) spectroscopy and found to be unique among described bacterial O-antigens. The O-antigen consists of hexasaccharide repeating units featuring a 4)-α-l-Fucp-(1-3)-ß-d-GlcpNAc-(1-4)-α-l-Fucp-(1-4)-ß-d-Glcp-(1- backbone, substituted with single residue side chains of α-d-Glcp and α-d-Quip3NAc linked to O-3 of the two fucose residues. The polysaccharide is partially O-acetylated on O-6 of the 4-substituted ß-Glcp residue.

Comprehensive analysis of the lysine acetylome in Aeromonas hydrophila reveals cross-talk between lysine acetylation and succinylation in LuxS.

Lysine acetylation and succinylation are both prevalent protein post-translational modifications (PTMs) in bacteria species, whereas the effect of the cross-talk between both PTMs on bacterial biological function remains largely unknown. Our previously study found lysine succinylated sites on proteins play important role on metabolic pathways in fish pathogenic Aeromonas hydrophila. A total of 3189 lysine-acetylation sites were further identified on 1013 proteins of this pathogen using LC-MS/MS in this study. Functional examination of these PTMs peptides showed associations with basal biological processes, especially metabolic pathways. Additionally, when comparing the obtained lysine acetylome to a previously obtained lysine succinylome, 1198 sites in a total of 547 proteins were found to be in common and associated with various metabolic pathways. As the autoinducer-2 (AI-2) synthase involved in quorum sensing of bacteria, the site-directed mutagenesis of LuxS at the K165 site was performed and revealed that the cross-talk between lysine acetylation and succinylation exerts an inverse influence on bacterial quorum sensing and on LuxS enzymatic activity. In summary, this study provides an in-depth A. hydrophila lysine acetylome profile and for the first time reveals the role of cross-talk between lysine acetylation and succinylation, and its potential impact on bacterial physiological functions.

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.

Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila.

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.

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.

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.

Chemical synthesis of the 4-amino-4,6-dideoxy-d-glucose containing pentasaccharide repeating unit of the O-specific polysaccharide from Aeromonas hydrophila strain K691 in the form of its 2-aminoethyl glycoside.

Chemical synthesis of the pentasaccharide repeating unit of the O-specific polysaccharide from Aeromonas hydrophilastrain K691 is reported. Synthesis of the pentasaccharide is accomplished by using a common disaccharide in sequence and finally attaching the rare sugar unit. The target structure was made in the form of its 2-aminoethyl glycoside which is essential for further glycconjugate formation. Stereoselective glycosylations were achieved by the activation of thioglycosides in the presence of H2SO4-silica in conjunction with N-iodosuccinimide.

Graphene-Based Steganographically Aptasensing System for Information Computing, Encryption and Hiding, Fluorescence Sensing and in Vivo Imaging of Fish Pathogens.

Inspired by information processing and communication of life based on complex molecular interactions, some artificial (bio)chemical systems have been developed for applications in molecular information processing or chemo/biosensing and imaging. However, little attention has been paid to simultaneously and comprehensively utilize the information computing, encoding, and molecular recognition capabilities of molecular-level systems (such as DNA-based systems) for multifunctional applications. Herein, a graphene-based steganographically aptasensing system was constructed for multifunctional application, which relies on specific molecular recognition and information encoding abilities of DNA aptamers ( Aeromonas hydrophila and Edwardsiella tarda-binding aptamers as models) and the selective adsorption and fluorescence quenching capacities of graphene oxide (GO). Although graphene-DNA systems have been widely used in biosensors and diagnostics, our proposed graphene-based aptasensing system can not only be utilized for fluorescence sensing and in vivo imaging of fish pathogens ( A. hydrophila and E. tarda), but can also function as a molecular-level logic computing system where the combination of matters (specific molecules or materials) as inputs produces the resulting product (matter level) or fluorescence (energy level) changes as two outputs. More importantly and interestingly, our graphene-based steganographically aptasensing system can also serve as a generally doubly cryptographic and steganographic system for sending different secret messages by using pathogen-binding DNA aptamers as information carriers, GO as a cover, and a pair of keys, that is, target pathogen as a public key, the encryption key used to encode or decode a message in DNA as a private key. Our study not only provides a novel nanobiosensing assay for rapid and effective sensing and in vivo imaging of fish pathogens, but also demonstrates a prototype of (bio)molecular steganography as an important and interesting extension direction of molecular information technology, which is helpful in probably promoting the development of multifunctional molecular-level devices or machines.

Proteomic characterization and discrimination of Aeromonas species recovered from meat and water samples with a spotlight on the antimicrobial resistance of Aeromonas hydrophila.

Aeromonas is recognized as a human pathogen following ingestion of contaminated food and water. One major problem in Aeromonas identification is that certain species are phenotypically very similar. The antimicrobial resistance is another significant challenge worldwide. We therefore aimed to use mass spectrometry technology for identification and discrimination of Aeromonas species and to screen the antimicrobial resistance of Aeromonas hydrophila (A. hydrophila). A total of 150 chicken meat and water samples were cultured, and then, the isolates were identified biochemically by the Vitek® 2 Compact system. Proteomic identification was performed by MALDI-TOF MS and confirmed by a microchannel fluidics electrophoresis assay. Principal component analysis (PCA) and single-peak analysis created by MALDI were also used to discriminate the Aeromonas species. The antimicrobial resistance of the A. hydrophila isolates was determined by Vitek® 2 AST cards. In total, 43 samples were positive for Aeromonas and comprised 22 A. hydrophila, 12 Aeromonas caviae (A. caviae), and 9 Aeromonas sobria (A. sobria) isolates. Thirty-nine out of 43 (90.69%) Aeromonas isolates were identified by the Vitek® 2 Compact system, whereas 100% of the Aeromonas isolates were correctly identified by MALDI-TOF MS with a score value ≥2.00. PCA successfully separated A. hydrophila, A. caviae and A. sobria isolates into two groups. Single-peak analysis revealed four discriminating peaks that separated A. hydrophila from A. caviae and A. sobria isolates. The resistance of A. hydrophila to antibiotics was 95.46% for ampicillin, 50% for cefotaxime, 45.45% for norfloxacin and pefloxacin, 36.36% for ceftazidime and ciprofloxacin, 31.81% for ofloxacin and 27.27% for nalidixic acid and tobramycin. In conclusion, chicken meat and water were tainted with Aeromonas spp., with a high occurrence of A. hydrophila. MALDI-TOF MS is a powerful technique for characterizing aeromonads at the genus and species levels. Future studies should investigate the resistance of A. hydrophila to various antimicrobial agents.

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.