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Abstract Zygaenoidea is a superfamily of lepidopterans containing many venomous species, including the Limacodidae (nettle caterpillars) and Megalopygidae (asp caterpillars). Venom proteomes have been recently documented for several species from each of these families, but further data are required to understand the evolution of venom in Zygaenoidea. In this study, we examined the ‘electric’ caterpillar from North-Eastern Australia, a limacodid caterpillar densely covered in venomous spines. We used DNA barcoding to identify this caterpillar as the larva of the moth Comana monomorpha (Turner, 1904). We report the clinical symptoms of C. monomorpha envenomation, which include acute pain, and erythema and oedema lasting for more than a week. Combining transcriptomics of venom spines with proteomics of venom harvested from the spine tips revealed a venom markedly different in composition from previously examined limacodid venoms that are rich in peptides. In contrast, the venom of C. monomorpha is rich in aerolysin-like proteins similar to those found in venoms of asp caterpillars (Megalopygidae). Consistent with this composition, the venom potently permeabilises sensory neurons and human neuroblastoma cells. This study highlights the diversity of venom composition in Limacodidae.

Zygaenoidea is a superfamily of lepidopterans containing many venomous species, including the Limacodidae (nettle caterpillars) and Megalopygidae (asp caterpillars). Venom proteomes have been recently documented for several species from each of these families, but further data are required to understand the evolution of venom in Zygaenoidea. In this study, we examined the 'electric' caterpillar from North-Eastern Australia, a limacodid caterpillar densely covered in venomous spines. We used DNA barcoding to identify this caterpillar as the larva of the moth Comana monomorpha (Turner, 1904). We report the clinical symptoms of C. monomorpha envenomation, which include acute pain, and erythema and oedema lasting for more than a week. Combining transcriptomics of venom spines with proteomics of venom harvested from the spine tips revealed a venom markedly different in composition from previously examined limacodid venoms that are rich in peptides. In contrast, the venom of C. monomorpha is rich in aerolysin-like proteins similar to those found in venoms of asp caterpillars (Megalopygidae). Consistent with this composition, the venom potently permeabilises sensory neurons and human neuroblastoma cells. This study highlights the diversity of venom composition in Limacodidae. [ABSTRACT FROM AUTHOR]

Intrinsically disordered protein regions (IDPRs) are pivotal in regulation of transcription and facilitation of signal transduction. Because of their multiple conformational states of structure, characterizing the highly flexible structures of IDPRs becomes challenging. Herein, we employed the wild‐type (WT) aerolysin nanopore as a real‐time biosensor for identification and monitoring of long peptides containing IDPRs. This sensor successfully identified three intrinsically disordered peptides, with the lengths up to 43 amino acids, by distinguishing the unique signatures of blockade current and duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptides are critical for peptide translocation, which suggests that mechanisms beyond mere volume exclusion. Furthermore, we were able to compare the conformational stabilities of various IDPRs by examining the detailed current traces of blockade events. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes. The nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery. [ABSTRACT FROM AUTHOR]

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Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site‐specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine‐based aminoacyl‐tRNA synthetase‐tRNA pair for a high yield of pore‐forming protein. Both molecular dynamics (MD) simulations and single‐molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches. [ABSTRACT FROM AUTHOR]

Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata. We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila, an opportunistic bacteria, causes several devastating diseases in humans and animals, particularly aquatic species. Antibiotics have been constrained by the rise of antibiotic resistance caused by drug overuse. Therefore, new strategies are required to prevent appropriate antibiotic inability from antibiotic-resistant strains. Aerolysin is essential for A. hydrophila pathogenesis and has been proposed as a potential target for inventing drugs with anti-virulence properties. It is a unique method of disease prevention in fish to block the quorum-sensing mechanism of A. hydrophila. In SEM analysis, the crude solvent extracts of both groundnut shells and black gram pods exhibited a reduction of aerolysin formation and biofilm matrix formation by blocking the QS in A. hydrophila. Morphological changes were identified in the extracts treated bacterial cells. Furthermore, in previous studies, 34 ligands were identified with potential antibacterial metabolites from agricultural wastes, groundnut shells, and black gram pods using a literature survey. Twelve potent metabolites showed interactions between aerolysin and metabolites during molecular docking analysis, in that H-Pyran-4-one-2,3 dihydro-3,5 dihydroxy-6-methyl (−5.3 kcal/mol) and 2-Hexyldecanoic acid (−5.2 kcal/mol) showed promising results with potential hydrogen bond interactions with aerolysin. These metabolites showed a better binding affinity with aerolysin for 100 ns in molecular simulation dynamics. These findings point to a novel strategy for developing drugs using metabolites from agricultural wastes that may be feasible pharmacological solutions for treating A. hydrophila infections for the betterment of aquaculture. [ABSTRACT FROM AUTHOR]

Simple Summary: Despite significant advancements in food safety and cleanliness, there is still a risk of food supply chain contamination. Fish are susceptible to infection from bacterial pathogens, especially Aeromonas spp. Aeromonas spp. are dangerous to people because they can spread illnesses like septic arthritis, gastroenteritis with diarrhea, skin and soft tissue infections, meningitis, and bacteremia through the consumption of infected fish. Therefore, the present study was conducted to detect the prevalence of, antibiotic resistance, virulence, and biofilm formation by Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt. In the current study, 11 isolates were confirmed as Aeromonas spp. and 4 isolates were confirmed as Aeromonas hydrophila using biochemical PCR assays. In addition, the antimicrobial resistance profile of the Aeromonas isolates was tested against 16 antibiotics, the result of which indicates the susceptibility of all isolates to imipenem followed by chloramphenicol, and a high multiple antibiotic resistance (MAR) index range between 0.142–0.642 was detected. In addition, it was illustrated that two isolates of the four identified A. hydrophila isolates were positive for the aer gene and that one isolate only had the hly gene. Additionally, biofilm formation was detected in two isolates from tilapia muscles and mugil viscera. Based on the information provided, control measures might be implemented to stop the high-risk contamination of a specific area and protect individuals from multidrug-resistant strains that might be transmitted through the food chain or incorrect handling. The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates' profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142–0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals. [ABSTRACT FROM AUTHOR]

A multi-technique approach to prove the preparation of poly(3,4-ethylenedioxythiophene/cucurbit[7]uril) pseudorotaxanes (PEDOT·CB7-PPs) is reported. Molecular docking simulation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) validate the complexation ability of the CB7 molecule towards 3,4-ethylenedioxythiophene (EDOT), which leads to the EDOT·CB7 inclusion complex. Oxidative polymerization of EDOT·CB7 enabled the synthesis of PEDOT·CB7-PPs. The water-soluble part of PEDOT·CB7-PPs was selected, freeze-dried, and chemically characterized. Furthermore, dynamic light scattering (DLS) has been used to study the particle size and z-potential (ZP-ζ) of PEDOT·CB7-PPs. The ZP-ζ value (35 mV) evidenced that the PEDOT·CB7-PPs formed stable water dispersion. By combining the emerging nanopore resistive pulse sensing technique (Np-RPS) and computational modeling, we identified strong interactions of PEDOT·CB7-PPs with the aerolysin (Ael) nanopore. PEDOT·CB7-PPs behave as positive charged species, and thus trans negative bias promotes its interactions with the Ael nanopore. The computational modeling results are fully consistent with the Np-RPS detection, which also reveals strong interactions between PEDOT·CB7-PPs and the Ael nanopore. With this study, we hope to provide new insights and a better understanding of the interactions between supramolecular complexes based on CB7 and biological entities, which is instrumental for future applications in the field of nanobiotechnology. [ABSTRACT FROM AUTHOR]

The overuse of antibiotics in aquaculture brings a range of problems. Therefore, novel approaches alternating antibiotics are urgently needed. Strategies targeting bacterial virulence have become one of the routes developing drugs against bacterial infections. Here, we found that pterostilbene (PSE) could inhibit the activity of aerolysin (AerA) and disrupt quorum sensing (QS) at sub-inhibitory concentrations. Molecular docking and dynamics simulation assays were performed to predict the binding mode and binding sites of AerA-PSE complex. Then we confirmed that PSE bound to the sites of Val189 and Ile302 and prevented AerA forming heptamer with transmembrane pores. In addition, PSE could protect to A549 cells and the channel catfish (Ictalurus punctatus) against AerA mediated cell injury and mortality infected with A. hydrophila. The findings demonstrated that targeting bacterial virulence could be a promising measure identifying agents against A. hydrophila infections. • Pterostilbene is an inhibitor of both aerolysin and quorum sensing of Aeromonas hydrophila. • Pterostilbene could reduce the pathogenicity of Aeromonas hydrophila at sub-inhibitory concentrations. • The main binding sites and mechanism of at pterostilbene against aerolysin was confirmed. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila (A. hydrophila) can cause a number of diseases in both human and animals. A. hydrophila-related infections in aquaculture cause severe economic losses every year throughout the world. The emergence of antibiotic resistance that is due to the abuse of antibiotics has limited the application of antibiotics. Thus, novel approaches are needed to combat with treatment failure of antibiotics caused by resistant bacterial strains. Aerolysin plays a critical role in the pathogenesis of A. hydrophila and has been considered as a novel target for developing drugs based on anti-virulence strategies. Here, we reported that luteolin, a natural product with no anti-A. hydrophila activity, could reduce aerolysin-induced hemolysis by inhibiting aerolysin activity. The binding mode was simulated by molecular docking and dynamics simulation. Then the main binding sites were confirmed by fluorescence quenching assays. We found that luteolin could hindered the formation of functional heptamer of aerolysin according to the results of the oligomerization assay. Moreover, luteolin could protect A549 cells from aerolysin mediated cell death and increase the survival rate of A. hydrophila-infected channel catfish. These findings suggest a novel approach to developing drugs fighting against A. hydrophila, and luteolin can be a promising drug candidate for treatment of A. hydrophila-associated infections.

The present study investigates pathogenicity of local Aeromonas hydrophila strains by molecular characterisation of two virulence factor genes: aerolysin (aerA) and haemolysin (Ahh1) using polymerase chain reaction (PCR) technique. Phenotypically identified presumptive Aeromonas isolates recovered from diseased Labeo rohita were genetically analysed using type-specific primers by amplifying 309 bp and 130 bp conserved regions of aerolysin and haemolysin genes respectively. The partial nucleotide sequences of aerA and Ahh1 were determined from representative strains in which aerA was confirmed in 75% isolates, whereas Ahh1 was confirmed in 50% isolates. The nucleotide blast results of the representative strains revealed close homology of 95% (aerolysin) and 97% (haemolysin) with published sequences.

Aeromonas hydrophila, an opportunistic aquatic pathogen widely spread in aquatic environments, is responsible for a number of infectious diseases in freshwater aquaculture. In addition, A. hydrophila can transmit from diseased fish to humans and results in health problems. The occurrence of antibiotic-resistant bacterial strains restricts the application of antibiotics and is responsible for failure of the treatment. Moreover, residues of antibiotics in aquatic products often threaten the quality and safety. Therefore, alternative strategies are called to deal with infections caused by antibiotic-resistant bacteria. Aerolysin, one of the most important virulence factors of A. hydrophila, is adopted as a unique anti-virulence target on the basis of the anti-virulence strategy to battling infections caused by A. hydrophila. Palmatine, an isoquinoline alkaloid from a variety of herbal medicines that showed no anti-A. hydrophila activity, could reduce hemolysis of the bacterium by decreasing aerolysin production. The results of the qPCR assay demonstrated that the transcription of the aerA gene was suppressed. Moreover, cell viability and in vivo study showed that palmatine treatment could decrease the pathogenicity of A. hydrophila both in vitro and in vivo. In summary, palmatine is a leading compound against A. hydrophila-associated infection in aquaculture by inhibiting the expression of aerolysin. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila, an opportunistic bacteria, causes several devastating diseases in humans and animals, particularly aquatic species. Antibiotics have been constrained by the rise of antibiotic resistance caused by drug overuse. Therefore, new strategies are required to prevent appropriate antibiotic inability from antibiotic-resistant strains. Aerolysin is essential for A. hydrophila pathogenesis and has been proposed as a potential target for inventing drugs with anti-virulence properties. It is a unique method of disease prevention in fish to block the quorum-sensing mechanism of A. hydrophila. In SEM analysis, the crude solvent extracts of both groundnut shells and black gram pods exhibited a reduction of aerolysin formation and biofilm matrix formation by blocking the QS in A. hydrophila. Morphological changes were identified in the extracts treated bacterial cells. Furthermore, in previous studies, 34 ligands were identified with potential antibacterial metabolites from agricultural wastes, groundnut shells, and black gram pods using a literature survey. Twelve potent metabolites showed interactions between aerolysin and metabolites during molecular docking analysis, in that H-Pyran-4-one-2,3 dihydro-3,5 dihydroxy-6-methyl (−5.3 kcal/mol) and 2-Hexyldecanoic acid (−5.2 kcal/mol) showed promising results with potential hydrogen bond interactions with aerolysin. These metabolites showed a better binding affinity with aerolysin for 100 ns in molecular simulation dynamics. These findings point to a novel strategy for developing drugs using metabolites from agricultural wastes that may be feasible pharmacological solutions for treating A. hydrophila infections for the betterment of aquaculture.

The fast-growing demand for aquatic products has led to the rapid development of aquaculture. However, diseases caused by bacterial pathogens result in severe economic losses all over the world. Although the introduction of antibiotics to aquaculture decreased the mortality of infectious diseases, the emergence of antibiotic resistance caused treatment failure. Therefore, drugs with novel strategies are needed for combatting infections caused by resistant bacterial strains. In the present study, aerolysin was identified as a target for developing drugs from natural compounds against Aeromonas hydrophila (A. hydrophila) infections. We found that polydatin without an inhibitory effect against A. hydrophila growth could decrease the hemolysis mediated by aerolysin. In both western blot and qPCR assays, the addition of polydatin decreased the production of aerolysin by downregulating the aerolysin encoding gene. Moreover, cell viability and animal studies found that polydatin could reduce the pathogenesis of A. hydrophila both in vitro and in vivo. Taken together, these findings provided a novel approach and candidate for treating resistant A. hydrophila infections in aquaculture.

Owing to their central roles in viral replication, main protease (Mpro) and papain-like protease (PLpro) are promising biomarkers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hence, the simultaneous sensing of Mpro and PLpro activities is a valuable approach for SARS-CoV-2 detection. Herein, we developed a dual response peptide probe for simultaneously monitoring the protease activities of Mpro and PLpro using a single aerolysin nanopore. The target proteases can specifically digest the probe at the corresponding cleavage sites, releasing the output peptide fragments composed of different amino acids. These fragments generated clearly distinguishable blocking currents in nanopore recording that can be assigned to each analyte, allowing accurate differentiation among multiple proteases. The approach enabled the sensitive detection of Mpro and PLpro activity without the need for any amplification or labeling. Furthermore, with excellent discrimination ability, the method can be applied to evaluate the activity of the two model proteases against a background of exhaled breath condensate. Importantly, since peptides with specific sequences serve as catalytic substrates of multiple enzymes, the proposed strategy would be applicable for accurate and multiplex quantification of different types of enzymes, which is critical for non-invasive early disease diagnosis. • A multiple detection strategy based on dual response peptide-assisted nanopore sensing is developed. • The limits of detection for both Mpro and PLpro reach the unprecedentedly low levels without labelling or amplification. • The nanopore sensor is successfully used to accurately identify and quantify the two proteases in real samples. [ABSTRACT FROM AUTHOR]

The genus Aeromonas is widely distributed in aquatic environments and is recognized as a potential human pathogen. Some Aeromonas species are able to cause a wide spectrum of diseases, mainly gastroenteritis, skin and soft-tissue infections, bacteremia, and sepsis. The aim of the current study was to determine the prevalence of Aeromonas spp. in raw fish markets and humans in Zagazig, Egypt; identify the factors that contribute to virulence; determine the isolates’ profile of antibiotic resistance; and to elucidate the ability of Aeromonas spp. to form biofilms. The examined samples included fish tissues and organs from tilapia (Oreochromis niloticus, n = 160) and mugil (Mugil cephalus, n = 105), and human skin swabs (n = 51) and fecal samples (n = 27). Based on biochemical and PCR assays, 11 isolates (3.2%) were confirmed as Aeromonas spp. and four isolates (1.2%) were confirmed as A. hydrophila. The virulence genes including haemolysin (hyl A) and aerolysin (aer) were detected using PCR in A. hydrophila in percentages of 25% and 50%, respectively. The antimicrobial resistance of Aeromonas spp. was assessed against 14 antibiotics comprising six classes. The resistance to cefixime (81.8%) and tobramycin (45.4%) was observed. The multiple antibiotic resistance (MAR) index ranged between 0.142–0.642 with 64.2% of the isolates having MAR values equal to 0.642. Biofilm formation capacity was assessed using a microtiter plate assay, and two isolates (18.1%) were classified as biofilm producers. This study establishes a baseline for monitoring and controlling the multidrug-resistant Aeromonas spp. and especially A. hydrophila in marine foods consumed in our country to protect humans and animals.

Aeromonas hydrophila is a bacterium associated with many diseases and disorders such as fin rot, skin ulcers and lethal hemorrhagic septicemia in fish. It bears several virulence factors including type III secretion system (T3SS), aerolysin, cytolytic enterotoxin and enzymes (e.g., hemolysins, lipase) that seem to play an important role in its pathogenesis. Detection of virulence markers by polymerase chain reaction (PCR) is a key procedure in defining the pathogenic ability of pathogenic bacteria and preparing a vaccine for its treatment. In this sense, this study was aimed to determine the frequency of virulence genes in isolates obtained from infected cultured carps in Khuzestan province. Out of 200 moribund carps with septicemic symptoms, 125 isolates were belonged to the motile aeromonads and 59 isolates were identified as A. hydrophila by biochemical methods. Finally, using PCR analysis, 31 isolates were identified as A. hydrophila. Five virulence genes were detected in these isolates including hemolysin, aerolysin, cytolytic enterotoxin and T3SS (aopB and ascV) by specific primers. Results showed that 23 (74.19%), 18 (58.06%), 16 (51.61%), 13 (41.63%) and 10 (32.25%) isolates possessed cytolytic enterotoxin, hemolysin, aerolysin, and T3SS genes, respectively. The results of the present study showed that among 31 isolates, only five isolates had all of dominant virulence genes. Thirteen other isolates had genotypes including hlyA+, aerA+, and act+. The remaining isolates had at least one virulence gene. This study showed that determination of the virulence genes by PCR can be a reliable method to identify a potential pathogenic Aeromonad strain. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila (A. hydrophila) can cause a number of diseases in both human and animals. A. hydrophila-related infections in aquaculture cause severe economic losses every year throughout the world. The emergence of antibiotic resistance that is due to the abuse of antibiotics has limited the application of antibiotics. Thus, novel approaches are needed to combat with treatment failure of antibiotics caused by resistant bacterial strains. Aerolysin plays a critical role in the pathogenesis of A. hydrophila and has been considered as a novel target for developing drugs based on anti-virulence strategies. Here, we reported that luteolin, a natural product with no anti-A. hydrophila activity, could reduce aerolysin-induced hemolysis by inhibiting aerolysin activity. The binding mode was simulated by molecular docking and dynamics simulation. Then the main binding sites were confirmed by fluorescence quenching assays. We found that luteolin could hindered the formation of functional heptamer of aerolysin according to the results of the oligomerization assay. Moreover, luteolin could protect A549 cells from aerolysin mediated cell death and increase the survival rate of A. hydrophila-infected channel catfish. These findings suggest a novel approach to developing drugs fighting against A. hydrophila, and luteolin can be a promising drug candidate for treatment of A. hydrophila-associated infections. [ABSTRACT FROM AUTHOR]

Abstract Background Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate. Results Relative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection. Conclusions Through these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.

Aeromonas hydrophila consider as the most common pathogenic bacteria which infect many farming fishes and due to many economical losses annually this bacteria found in fresh, brackish waters and ponds. The present study conducted to determine the most reliable method for the diagnosis of A. hydrophila from common carp (Cyprinus carpio), in Agriculture college fish farmduring a farming (May 2017 to October 2018).Results of the present study showed that 60 bacterial samples out of 115 bacterial samples were gram-negative, however 40 samples were motile and only 29 samples showed positive hemolysis on blood agar. Result of molecular diagnosis showed that only 18 bacterial samples have Aerolysin gene (1482 bp).

The functional characterization of an Amaranthus hypochondriacus Natterin-4-Like-1 gene (AhN4L-1) coding for an unknown function protein characterized by the presence of an aerolysin-like pore-forming domain in addition to two amaranthin-like agglutinin domains is herewith described. Natterin and nattering-like proteins have been amply described in the animal kingdom. However, the role of nattering-like proteins in plants is practically unknown. The results described in this study, obtained from gene expression data in grain amaranth and from AhN4L-1-overexpressing Arabidopsis thaliana plants indicated that this gene was strongly induced by several biotic and abiotic conditions in grain amaranth, whereas data obtained from the overexpressing Arabidopsis plants further supported the defensive function of this gene, mostly against bacterial and fungal plant pathogens. GUS and GFP AhN4L-1 localization in roots tips, leaf stomata, stamens and pistils also suggested a defensive function in these organs, although its participation in flowering processes, such as self-incompatibility and abscission, is also possible. However, contrary to expectations, the overexpression of this gene negatively affected the vegetative and reproductive growth of the transgenic plants, which also showed no increased tolerance to salinity and water-deficit stress. The latter despite the maintenance of significantly higher chlorophyll levels and photosynthetic parameters under intense salinity stress. These results are discussed in the context of the physiological roles known to be played by related lectins and AB proteins in plants.

Subtilosin A, a cyclic peptide from Bacillus subtilis is known for its antimicrobial activity against a diverse range of bacteria. Herein, we report the specific interaction between subtilosin A against virulent proteins of Aeromonas hydrophila through in silico analysis. Aeromonas toxic proteins such as aerolysin and hemolysin were selected from the non-redundant database. The hemolysin protein was designed by homology modelling tool, and it was validated using Ramachandran plot. Then subtilosin A and target toxin proteins were energy minimized for further docking study. The whole docking experiments were done using antibody mode in Cluspro. Subtilosin A building an active interaction with Aeromonas toxins through H-bonds and protein–protein docking analysis revealed that the hemolysin has 6 H-bond interaction towards the antimicrobial target protein subtilosin A than aerolysin, which has 9 H-bonds. The most favourable interacting residues of subtilosin A are Thr6, Cys13, Ile19, Pro20, Asp21, Phe22, Glu23 and Gly35 involving in the strong H-bond formation and proceeds to inhibition of toxin. Hence, the study confirmed that the subtilosin A has more antimicrobial activity to inhibit the Aeromonas toxins by interacting with their binding site residues for preventing extracellular cleavage. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila, an opportunistic aquatic pathogen widely spread in aquatic environments, is responsible for a number of infectious diseases in freshwater aquaculture. In addition, A. hydrophila can transmit from diseased fish to humans and results in health problems. The occurrence of antibiotic-resistant bacterial strains restricts the application of antibiotics and is responsible for failure of the treatment. Moreover, residues of antibiotics in aquatic products often threaten the quality and safety. Therefore, alternative strategies are called to deal with infections caused by antibiotic-resistant bacteria. Aerolysin, one of the most important virulence factors of A. hydrophila, is adopted as a unique anti-virulence target on the basis of the anti-virulence strategy to battling infections caused by A. hydrophila. Palmatine, an isoquinoline alkaloid from a variety of herbal medicines that showed no anti-A. hydrophila activity, could reduce hemolysis of the bacterium by decreasing aerolysin production. The results of the qPCR assay demonstrated that the transcription of the aerA gene was suppressed. Moreover, cell viability and in vivo study showed that palmatine treatment could decrease the pathogenicity of A. hydrophila both in vitro and in vivo. In summary, palmatine is a leading compound against A. hydrophila-associated infection in aquaculture by inhibiting the expression of aerolysin.

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs. Liu et al. construct a knock out cell library of genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored protein (GPI-APs) from HEK-293 cells and analyzed the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C. Furthermore, the authors also identify structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. [ABSTRACT FROM AUTHOR]

Vibrio splendidus is one of the main pathogens caused diseases with a diversity of marine cultured animals, especially the skin ulcer syndrome in Apostichopus japonicus. However, limited virulence factors have been identified in V. splendidus. In this study, one aerA Vs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerA Vs was analyzed. Western blotting was used to study of the secretion pathway of proaerolysin, and it showed that the proaerolysin was secreted via both outer membrane vehicles and classical secretion pathways. Since no active protein of aerolysin was obtained, one aerolysin surface displayed bacterium DH5α/pAT- aerA was constructed, and its haemolytic activity and virulence were determined. The results showed that the AerA Vs displayed on the surface showed obvious haemolytic activity and cytotoxic to the coelomocyte of A. japonicus. Artificial immerse infection separately using the DH5α/pAT or DH5α/pAT- aerA was conducted. The result showed that the mortality percent of sea cucumber A. japonicus challenged with DH5α/pAT- aerA was 38.89 % higher than that challenged with the control strain DH5α/pAT, and earlier death occurred. Combined all the results indicates that aerolysin with the haemolytic activity and cytotoxic activity is a virulence factor of V. splendidus. • One aerA Vs gene, coding aerolysin, was cloned from a pathogenic V. splendidus. • The DH5α/pAT- aerA with AerA Vs displayed on the cell surface was constructed. • AerA Vs possessed haemolytic and cytotoxic activities contributing to virulence. [ABSTRACT FROM AUTHOR]

The intracellular protozoan parasite, Theileria annulata, induces uncontrolled proliferation and transformation in bovine B lymphocytes and monocytes in blood circulation andlymph nodes of host cells. This uncontrolled replication happens in the macroschizont stage of the life cycle of the parasites. The development of a rapid and efficient technique is likely to necessitate for isolation of purified schizonts from host cells. This is necessary for the isolation of highly purified RNA, protein or glycoproteins of schizonts from host cells. This study aimed to isolate the purified schizont based on the aerolysin–nocodazole technique. Aerolysin which was purified from gram-negative pathogen aeromonas hydrophila has an ability to form discrete channels and unstable eukaryotic cell membranes in 0C• and low concentration. Nocodazole used for parasite separation from the microtubule network of infected lymphocytes and monocytes. In purified schizonts, no large nucleus of host cells visualized in giemsa and DAPI staining. The isolated schizonts were free and intact from host cells. Intact T.annulata schizonts obtained from this study are suitable for purification of RNAs, proteins, glycolipids, and glycoproteins of schizonts free of host cell debris.

Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family. Twenty-three biomphalysin genes were identified. All are expressed and exhibited a tissue-specific expression pattern. An in silico structural analysis was performed to highlight the central role played by two distinct domains i) a large lobe involved in the lytic function of these snail toxins which constrained their evolution and ii) a small lobe which is structurally variable between biomphalysin toxins and that matched to various functional domains involved in moiety recognition of targets cells. A functional approach suggests that the repertoire of biomphalysins that bind to pathogens, depends on the type of pathogen encountered. These results underline a neo-and sub-functionalization of the biomphalysin toxins, which have the potential to increase the range of effectors in the snail’s immune arsenal.

Aerolysins initially characterized as virulence factors in bacteria are increasingly found in massive genome and transcriptome sequencing data from metazoans. Horizontal gene transfer has been demonstrated as the main way of aerolysin-related toxins acquisition in metazoans. However, only few studies have focused on their potential biological functions in such organisms. Herein, we present an extensive characterization of a multigene family encoding aerolysins - named biomphalysin - in Biomphalaria glabrata snail, the intermediate host of the trematode Schistosoma mansoni. Our results highlight that duplication and domestication of an acquired bacterial toxin gene in the snail genome result in the acquisition of a novel and diversified toxin family. Twenty-three biomphalysin genes were identified. All are expressed and exhibited a tissue-specific expression pattern. An in silico structural analysis was performed to highlight the central role played by two distinct domains i) a large lobe involved in the lytic function of these snail toxins which constrained their evolution and ii) a small lobe which is structurally variable between biomphalysin toxins and that matched to various functional domains involved in moiety recognition of targets cells. A functional approach suggests that the repertoire of biomphalysins that bind to pathogens, depends on the type of pathogen encountered. These results underline a neo-and sub-functionalization of the biomphalysin toxins, which have the potential to increase the range of effectors in the snail's immune arsenal. [ABSTRACT FROM AUTHOR]

Secretory pore-forming proteins (PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs (af-PFPs) and trefoil factors (TFFs). As a toad af-PFP, BmALP1 can be reversibly regulated between active and inactive forms, with its paralog BmALP3 acting as a negative regulator. BmALP1 interacts with BmTFF3 to form a cellular active complex called βγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of βγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence, we hereby propose a secretory endolysosome channel (SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with βγCAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms. [ABSTRACT FROM AUTHOR]

The present study was intended to elucidate the genomic basis of antibiotic resistance and hyper-virulence of the fish pathogen Aeromonas veronii XhG1.2 characterized in our previous work. The identity of XhG1.2 was confirmed through 16S rDNA sequence analysis and whole genome sequence analysis. The top-hit species distribution analysis of XhG1.2 sequence data revealed major hits against the Aeromonas veronii. The identification of virulence genes using the VFDB showed the genome of XhG1.2 to have the genes coding for the virulence factors viz. aerolysin, RtxA, T2SS, T3SS and T6SS. The presence of antibiotic resistance predicted through the CARD database analysis showed it to have the Ceph A3, OXA-12, ade F and pulvomycin resistance genes. By the phylogenetic and comparative genomic analysis, A. veronii species were found to have genes for toxin production. This also confirmed the pathogenicity and drug resistance of A. veronii XhG1.2 and also its potential to cause disease in diverse ornamental fishes. • The evolution of antibiotic resistance in aquatic bacteria Aeromonas veronii XhG1.2. • Genomic DNA sequencing and 16S rDNA analysis. • COG analysis showed 304 general function genes &285 amino acid transport and metabolism genes. • Major virulence factors predicted as aerolysin, RtxA, T2SS, T3SS, T6SS, adherence and motility. • Major antibiotic resistance genes found are Cph A3, OXA-12, aed F and Pulvomycin. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hydrobiology is the property of Editorial Department of Journal of Hydrobiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aeromonas species are highly distributed in the world and causative agent for fish, animal and human diseases. The study concluded 255 fecal sampler (168 sample were collected from patient suffering from diarrhea and 82 sample from apparently healthy). The Aeromonas hydrophila isolates were identified depended on culture media and biochemical methods for I field A.hydrophila; the PCR technique used to detection thewhole forming Toxin (aerolysin) gene. The results revealed that 23 isolates of A. hydrophila were identified from diarrheal patients, while 18 isolates from apparently healthy. Aerolysin gene was detected in 9 isolates only. To determine the in vivo pathogenicity of A. hydrophila, forty two mice were used to detect LD50 value, then determined the histological changes of organs including intestine and liver were obtained for re-isolation and histopathological examination. Lethal dose of also demonstrated in mice was 0.9×1000000000. The microscopic examination of histopathological sections of intestine of infected mice after LD50 experiment showed that preserver of hyperplasia of lymphoid tissue with thickening in villi, congestion of central vein of liver, mild inflammation in periportal area, fatty changes and parenchymal cell of liver. [ABSTRACT FROM AUTHOR]

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Aeromonas hydrophila is one of common bacterial disease in aquatic animals and its outbreak cause to decrease of aquatic production. Aeromonas disease is due to a protein toxin, aerolysin that exported by Aeromonas hydrophila. This protein toxin forms channels on target cells membrane, disrupting normal activities and cause to destruction and death of them. Aerolysin toxic protein is secreted by expression of the aerolysin gene. Aerolysin gene is 1482 bp and it’s an important and stable molecular marker for detecting possible of Aeromonas hydrophila infection. The PCR technique was used to study of this molecular marker. Different antibiotics used to control and treat of Aeromonas disease and tetracycline is one of the importances of them. Antibacterial mechanism of tetracycline is interference in the translation of aerolysin gene and therefore inhibition of protein synthesis in ribosome of bacteria, therefore they condemn dying. Knowledge of the antimicrobial mechanism of antibiotics can play an important role in finding suitable alternative materials for these synthetic drugs.

There is a current need to develop methods for the sensitive detection of peptide biomarkers in complex mixtures of molecules, such as biofluids, to enable early disease detection. Moreover, to our knowledge, there is currently no detection method capable of identifying the different conformations of a peptide biomarker differing by a single amino acid. Single-molecule nanopore sensing promises to provide this level of resolution. In order to be able to identify these differences in a biofluid such as serum, it is necessary to carefully characterize electrical parameters to obtain specific signatures of each biomarker population observed. We are interested here in a family of peptide biomarkers, kinins such as bradykinin and des-Arg 9 bradykinin, that are involved in many disabling pathologies (allergy, asthma, angioedema, sepsis, or cancer). We show the proof of concept for direct identification of these biomarkers in serum at the single-molecule level using a protein nanopore. Each peptide exhibits two unique electrical signatures attributed to specific conformations in bulk. The same signatures are found in serum, allowing their discrimination and identification in a complex mixture such as biofluid. To extend the utility of our experimental results, we developed a principal component analysis approach to define the most relevant electrical parameters for their identification. Finally, we used semisupervised classification to assign each event type to a specific biomarker at physiological serum concentration. In the future, single-molecule scale analysis of peptide biomarkers using a powerful nanopore coupled with machine learning will facilitate the identification and quantification of other clinically relevant biomarkers from biofluids.

Aeromonas hydrophila (A. hydrophila) is an opportunistic bacterial pathogen widely distributed in the environments, particular aquatic environment. The pathogen can cause a range of infections in both human and animals including fishes. However, the application of antibiotics in treatment of A. hydrophila infections leads to the emergence of resistant strains. Consequently, new approaches need to be developed in fighting this pathogen. Aerolysin, the chief virulence factor produced by pathogenic A. hydrophila strains has been employed as target identifying new drugs. In our present study, we found that morin, a flavonoid without anti-bacterial activity isolated from traditional Chinese medicine, could directly inhibit the hemolytic activity of aerolysin. To determine the binding sites and the action of mechanism of morin against AerA, several assays were performed. Ser36, Pro347, and Arg356 were identified as the main binding sites affecting the conformation of AerA and resulted in block of the heptameric formation. Moreover, morin could protect Vero cells from cell injury mediated by aerolysin. In vivo study showed that morin could provide a protection to channel catfish against A. hydrophila infection. These results demonstrated that morin could be developed as a promising candidate for the treatment of A. hydrophila infections by decreasing the pathogenesis of A. hydrophila.

Aeromonas hydrophila was isolated from an African black-footed penguin (Spheniscus demersus) that died while in zoo captivity. At necropsy, the virulence of A. hydrophila appeared to be enhanced by stress, so was assessed in the presence of in vitro and in vivo stressors and serial passaging in mice. Virulence genes from the isolate were amplified by PCR. In vitro assays were conducted to test the hemolytic activity, cytotoxicity, and effect of stress hormones on A. hydrophila virulence. In vivo assays were conducted to test the stress effect on mortality of A. hydrophila-infected mice and virulence in mice. Two virulence genes coding for hemolysin (ahh1) and aerolysin (aerA) were detected, and the cytotoxic potential of the isolate was demonstrated in baby hamster kidney and Vero cells. Some or all mice inoculated with A. hydrophila and exposed to stress hormones (epinephrine and norepinephrine) or low temperature died, while mice inoculated with A. hydrophila and exposed to fasting or agitation stressors or no stressors survived. We concluded that stress can be fatal in mice experimentally infected with A. hydrophila and that serial passaging in mice dramatically enhances the virulence of A. hydrophila.

Nanopore biosensors, as an extremely sensitive tool, are widely applied to single‐molecule analysis. Compared with the commonly used α‐hemolysin, aerolysin nanopore possess a narrower channel, which distributes the abundant charged residues in the lumen. We replaced the commonly used potassium cations (K+) with tetramethylammonium cations (TMA+) to explore the cation effects. Experimental results demonstrate that the blockage of oligonucleotides in the TMA+ system is distinctly more considerable than that in K+ system. In particular, the blockade currents of Poly(dA)5 are very closed to 0 pA. In addition, the capture rate of oligonucleotides is distinctly increased, and it is speculated that amphipathic TMA+ can enhance the affinity of aerolysin nanopore for oligonucleotides. Furthermore, the estimated effective charges (Zinside) of oligonucleotides inside aerolysin nanopore in two solutions suggest that the screening of K+ on Poly(dA)3, Poly(dA)4, and Poly(dA)5 is more effective than that for TMA+. This report deepens our understanding of the effects of cations in electrolyte. [ABSTRACT FROM AUTHOR]

Aerolysin (aer) is one of the most important and abundant virulence factors in the infection of fish by Aeromonas veronii. A comprehensive study on the molecular characterization and pathogenicity of the aer gene from 34 A. veronii isolates from diseased carp and catfish was carried out and its interactome was analysed to observe the functional correlations between aer and other proteins within the A. veronii network. The PCR‐based amplification of aer from the 34 isolates of A. veronii showed more aer‐positive isolates from catfish with a high pathogenic potential in the in vivo challenge test than the carp fish. The analysis of aer gene sequence from challenged fish revealed significant sequence divergence according to the types and geographical distribution of the fish. The networking analysis of aer from the model A. veronii B565 revealed histidine kinase (cheA) as the most functional interacting partner. The study of the interaction between aer from the experimental A. veronii and cheA demonstrated that the A chain of cheA plays a more important role than the corresponding B chain during contact, and a linker sequence of 15 residues controlled the entire interaction process. Therefore, cheA could be an excellent drug target for controlling A. veronii infection of fish. [ABSTRACT FROM AUTHOR]

The protein acyl transferase ZDHHC5 was recently proposed to regulate trafficking in the endocytic pathway. Therefore, we explored the function of this enzyme in controlling the action of bacterial toxins. We found that ZDHHC5 activity is required for two very different toxins: the anthrax lethal toxin and the pore-forming toxin aerolysin. Both of these toxins have precursor forms, the protoxins, which can use the proprotein convertases Furin and PC7 for activation. We show that ZDHHC5 indeed affects the processing of the protoxins to their active forms. We found that Furin and PC7 can both be S-palmitoylated and are substrates of ZDHHC5. The impact of ZDHHC5 on Furin/PC7-mediated anthrax toxin cleavage is dual, having an indirect and a direct component. First, ZDHHC5 affects the homeostasis and trafficking of a subset of cellular proteins, including Furin and PC7, presumably by affecting the endocytic/recycling pathway. Second, while not inhibiting the protease activity per se, ZDHHC5-mediated Furin/PC7 palmitoylation is required for the cleavage of the anthrax toxin. Finally, we show that palmitoylation of Furin and PC7 promotes their association with plasma membrane microdomains. Both the receptor-bound toxin and the convertases are of very low abundance at the cell surface. Their encounter is unlikely on reasonable time scales. This work indicates that palmitoylation drives their encounter in specific domains, allowing processing and thereby intoxication of the cell. [ABSTRACT FROM AUTHOR]

The aerolysin nanopore has a high sensitivity for molecular sensing, owing to its super‐narrow diameter and highly charged lumen structure. Here, taking advantages of its high sensitivity, we investigated peptides with comparable size and mass but different charges. Based on the blockade current and duration time, we found that the charge distribution at the entrance of the aerolysin nanopore governs the capture of peptides. Moreover, peptides with the same charge density but different lengths were measured by the aerolysin nanopore. In comparison to the short peptides, longer peptides gave a larger blockade current and a longer duration time. Our findings demonstrate that both the charge and length of peptides affect the translocation process simultaneously, which improve our understanding of peptides' translocation through the aerolysin nanopore and suggest that aerolysin is an excellent candidate for peptides/protein sequencing. Lift the blockade: Three negatively charged peptides with different lengths are driven to translocate the aerolysin nanopore inducing three typical current blockades, which perform differences in current amplitude and duration. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila (A. hydrophila) is an opportunistic bacterial pathogen widely distributed in the environments, particular aquatic environment. The pathogen can cause a range of infections in both human and animals including fishes. However, the application of antibiotics in treatment of A. hydrophila infections leads to the emergence of resistant strains. Consequently, new approaches need to be developed in fighting this pathogen. Aerolysin, the chief virulence factor produced by pathogenic A. hydrophila strains has been employed as target identifying new drugs. In our present study, we found that morin, a flavonoid without anti-bacterial activity isolated from traditional Chinese medicine, could directly inhibit the hemolytic activity of aerolysin. To determine the binding sites and the action of mechanism of morin against AerA, several assays were performed. Ser36, Pro347, and Arg356 were identified as the main binding sites affecting the conformation of AerA and resulted in block of the heptameric formation. Moreover, morin could protect Vero cells from cell injury mediated by aerolysin. In vivo study showed that morin could provide a protection to channel catfish against A. hydrophila infection. These results demonstrated that morin could be developed as a promising candidate for the treatment of A. hydrophila infections by decreasing the pathogenesis of A. hydrophila. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is an opportunistic pathogen responsible for a number of diseases in freshwater farming. Moreover, the bacterium has been identified as a zoonotic pathogen that threatens human health. Antibiotics are widely used for treatments of infectious diseases in aquaculture. However, the abuse of antibiotics has led to the emergence of antimicrobial resistant strains. Thus, novel strategies are required against resistant A. hydrophila strains. The quorum sensing (QS) system, involved in virulence factor production and biofilm formation, is a promising target in identifying novel drugs against A. hydrophila infections. In this study, we found that thymol, at sub-inhibitory concentrations, could significantly reduce the production of aerolysin and biofilm formation by inhibiting the transcription of genes aerA, ahyI, and ahyR. These results indicate that thymol inhibits the quorum sensing system. The protective effects of thymol against A. hydrophila mediated cell injury were determined by live/dead assay and lactate dehydrogenase (LDH) release assay. Moreover, the in vivo study showed that thymol could significantly decrease the mortality of channel catfish infected with A. hydrophila. Taken together, these findings demonstrate that thymol could be chosen as a phytotherapeutic candidate for inhibiting quorum sensing system-mediated aerolysin production and biofilm formation in A. hydrophila.

This work aimed to investigate the virulence factors and antibiotic resistance of A. hydrophila isolated from different sources in Damietta Governorate, Egypt. The samples comprised of tilapia (Oreochromis niloticus, n=150), mugil (Mugil cephalus, n=144), human stool (n=102) and fish sellers hand swabs (n=85). A total of 25 A. hydrophila isolates were recovered and molecularly confirmed, of which, 2.7% were from tilapia muscles, 2% from tilapia viscera, 6.3% from mugil viscera and 8.8% from stool samples. No isolates were recovered from mugil muscles and hand swabs. Phenotypic determination of heamolysis, lipolysis, proteolysis and gelatinase activity revealed that 60%, 56%, 60% and 100% were positive, respectively. Molecular identification of haemolysin (hly), aerolysin (aer), lipase (lip) and cytotoxic enterotoxins (act) virulence associated genes showed that 28%, 68%, 28% and 16% of the examined isolates were positive, respectively. Antimicrobial susceptibility of the isolates to 17 antibiotics was determined, the most resistance pattern was observed for cefixime (88%), while, all of the isolates were susceptible to imipenem. This study revealed that A. hydrophila isolated from fish and humans showed several virulence factors and exhibit a wide range of antibiotic resistance which is considered a public health hazard as well as. [ABSTRACT FROM AUTHOR]

Summary: Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ‐free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono‐infection, but also A. hydrophila NJ‐1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii‐targeted treatments in future efforts against MAS. [ABSTRACT FROM AUTHOR]

Copyright of Pesquisa Veterinaria Brasileira is the property of Colegio Brasileiro de Patologia Animal - CBPA and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit fish and tilapia, and an invertebrate (abalone) in Oman during December 2011-May 2012. The cytotoxic enterotoxin ast gene was found widely distributed among the isolates; aerolysin-like protein (act) and the flagellin structural gene lafA less so; and the nuclease gene (nuc) not at all. However, there was not any evidence of pathogenicity among the isolates when tested in laboratory-based experiments using rainbow trout and Nile tilapia. Therefore, the risk of the pathogen to fish in Oman is unclear. [ABSTRACT FROM AUTHOR]