Phylogeny, envenomation syndrome, and membrane permeabilising venom produced by Australia's electric caterpillar Comana monomorpha.

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.

Detection of intrinsically disordered peptides by biological nanopore.

Intrinsically disordered protein regions (IDPRs) are pivotal in the regulation of transcription and the 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 the 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 leveraging the unique signatures of blockade current and translocation duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptide are critical for peptide translocation, suggesting that mechanisms beyond mere volume exclusion are at play. Furthermore, by examining the detailed current traces of blockade events, we were able to compare the conformational stabilities of various IDPRs. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes under the confined space. This nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery.

Cross-sectional analysis of risk factors associated with Mugil cephalus in retail fish markets concerning methicillin-resistant Staphylococcus aureus and Aeromonas hydrophila.

Introduction: Aeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated. Methods: In a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers' public health implications were assessed via their response to a descriptive questionnaire. Results: S. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category. Conclusion: The pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health.

Cloning and characterization of an aerolysin gene from a marine pathogen Vibrio splendidus.

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 aerAVs gene coding an aerolysin of V. splendidus was cloned and conditionally expressed in Escherichia coli. The haemolytic activity of the recombinant AerAVs 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 AerAVs 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.

Identification of Conformational Variants for Bradykinin Biomarker Peptides from a Biofluid Using a Nanopore and Machine Learning.

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-Arg9 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.

Caenorhabditis elegans LIN-24, a homolog of bacterial pore-forming toxin, protects the host from microbial infection.

Aerolysin-like pore-forming protein (af-PFP) superfamily members are double-edge swords that assist the bacterial infection but shied bacteria from the host by various mechanisms in some species including the toad Bombina maxima and zebrafish. While members of this family are widely expressed in all kingdoms, especially non-bacteria species, it remains unclear whether their anti-bacterial function is conserved. LIN-24 is an af-PFP that is constitutively expressed throughout the Caenorhabditis elegans lifespan. Here, we observed that LIN-24 knockdown reduced the maximum lifespan of worms. RNA-seq analysis identified 323 differentially expressed genes (DEGs) post-LIN-24 knockdown that were enriched in "immune response" and "lysosome pathway," suggesting a possible role for LIN-24 in resisting microbial infection. In line with this, we found that Pseudomonas aeruginosa 14 (PA14) infection induced LIN-24 expression, and that survival after PA14 infection was significantly reduced by LIN-24 knockdown. In contrast, LIN-24 overexpression (LIN-24-OE) conferred protection against PA14 infection, with worms showing longer survival time and reduced bacterial load. Weighted gene co-expression network analysis of LIN-24-OE worms showed that the highest correlation module was enriched in factors related to immunity and the defense response. Finally, by predicting transcription factors from RNA-seq data and knocking down candidate transcription factors in LIN-24-OE worms, we revealed that LIN-24 may protect worms against bacterial infection by stimulating DAF-16-mediated immune responses. These findings agree with our previous studies showing an anti-microbial role for the amphibian-derived af-PFP complex ßγ-CAT, suggesting that af-PFPs may play a conserved role in combatting microbial infections. Further research is needed to determine the roles this protein family plays in other physio-pathological processes, such as metabolism, longevity, and aging.

Horizontal gene transfer underlies the painful stings of asp caterpillars (Lepidoptera: Megalopygidae).

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.

ß Pore-forming Protein-based Evolutionary Divergence of Gnathostomata from Agnatha.

INTRODUCTION: The first vertebrates were jawless fish, or Agnatha, whose evolution diverged into jawed fish, or Gnathostomes, around 550 million years ago. METHODS: In this study, we investigated ß PFT proteins' evolutionary divergence of lamprey immune protein from Agnatha, reportedly possessing anti-cancer activity, into Dln1 protein from Gnathostomes. Both proteins showed structural and functional divergence, and shared evolutionary origin. Primary, secondary and tertiary sequences were compared to discover functional domains and conserved motifs in order to study the evolution of these two proteins. The structural and functional information relevant to evolutionary divergence was revealed using hydrophobic cluster analysis. RESULTS: The findings demonstrate that two membrane proteins with only a small degree of sequence identity can have remarkably similar hydropathy profiles, pointing towards conserved and similar global structures. When facing the lipid bilayer or lining the pore lumen, the two proteins' aerolysin domains' corresponding residues displayed a similar and largely conserved pattern. Aerolysin-like proteins from different species can be identified using a fingerprint created by PIPSA analysis of the pore-forming protein. CONCLUSION: We were able to fully understand the mechanism of action during pore formation through structural studies of these proteins.

Evaluating Biofilm Inhibitory Potential in Fish Pathogen, Aeromonas hydrophila by Agricultural Waste Extracts and Assessment of Aerolysin Inhibitors Using In Silico Approach.

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.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

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.

Observing Confined Local Oxygen-induced Reversible Thiol/Disulfide Cycle with a Protein Nanopore.

Disulfide bonds play an important role in thiol-based redox regulation. However, owing to the lack of analytical tools, little is known about how local O2 mediates the reversible thiol/disulfide cycle under protein confinement. In this study, a protein-nanopore inside a glove box is used to control local O2 for single-molecule reaction, as well as a single-molecule sensor for real-time monitoring of the reversible thiol/disulfide cycle. The results demonstrate that the local O2 molecules in protein nanopores could facilitate the redox cycle of disulfide formation and cleavage by promoting a higher fraction of effective reactant collisions owing to nanoconfinement. Further kinetic calculations indicate that the negatively charged residues near reactive sites facilitate proton-involved oxygen-induced disulfide cleavage under protein confinement. The unexpectedly strong oxidation ability of confined local O2 may play an essential role in cellular redox signaling and enzyme reactions.

Development of a surface plasmon resonance (SPR) assay for rapid detection of Aeromonas hydrophila.

Aquaculture plays an increasingly important if not critical role in the current and future world food supply. Aeromonas hydrophila, a heterotrophic, Gram-negative, bacterium found in fresh or brackish water in warm climates poses a serious threat to the aquaculture industry in many areas, causing significant economic losses. Rapid, portable detection methods of A. hydrophila are needed for its effective control and mitigation. We have developed a surface plasmon resonance (SPR) technique to detect PCR (polymerase chain reaction) products that can replace agarose gel electrophoresis, or otherwise provide an alternative to costlier and more complicated real-time, fluorescence-based detection. The SPR method provides sensitivity comparable to gel electrophoresis, while reducing labor, cross-contamination, and test time, and employs simpler instrumentation with lower cost than real-time PCR.

Antibiotic Resistance, Virulence Gene Detection, and Biofilm Formation in Aeromonas spp. Isolated from Fish and Humans in Egypt.

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.

Interaction of Clostridium perfringens Epsilon Toxin with the Plasma Membrane: The Role of Amino Acids Y42, Y43 and H162.

Clostridium perfringens epsilon toxin (Etx) is a pore forming toxin that causes enterotoxaemia in ruminants and may be a cause of multiple sclerosis in humans. To date, most in vitro studies of Etx have used the Madin-Darby canine kidney (MDCK) cell line. However, studies using Chinese hamster ovary (CHO) cells engineered to express the putative Etx receptor, myelin and lymphocyte protein (MAL), suggest that amino acids important for Etx activity differ between species. In this study, we investigated the role of amino acids Y42, Y43 and H162, previously identified as important in Etx activity towards MDCK cells, in Etx activity towards CHO-human MAL (CHO-hMAL) cells, human red blood cells (hRBCs) and synthetic bilayers using site-directed mutants of Etx. We show that in CHO-hMAL cells Y42 is critical for Etx binding and not Y43 as in MDCK cells, indicating that surface exposed tyrosine residues in the receptor binding domain of Etx impact efficiency of cell binding to MAL-expressing cells in a species-specific manner. We also show that Etx mutant H162A was unable to lyse CHO-hMAL cells, lysed hRBCs, whilst it was able to form pores in synthetic bilayers, providing evidence of the complexity of Etx pore formation in different lipid environments.

Aerolysin gene characterization and antimicrobial resistance profile of Aeromonas hydrophila isolated from milkfish (Chanos chanos) in Gresik, Indonesia.

Background and Aim: Motile Aeromonas septicemia is a crucial disease in freshwater fish. Aeromonas hydrophila is a disease agent associated with sporadic fish mortality, food safety, and public health. This study aimed to estimate the prevalence and the presence of the aerolysin gene and antimicrobial resistance profile of A. hydrophila isolated from milkfish in Gresik, Indonesia. Materials and Methods: A total of 153 milkfish gill samples were collected from 16 locations in Gresik and then cultured and identified using biochemical tests. The aerolysin gene was investigated using a polymerase chain reaction, and antimicrobial resistance profiles of the recovered isolates were investigated. Results: Of the 153 examined samples, 35 (22.9%) were confirmed positive for A. hydrophila and 22 (62.9%) presented the aerolysin gene. The recovered isolates were resistant to the following antibiotics: Amoxicillin (62.9%), tetracycline (60%), streptomycin (54.3%), cefotaxime (51.4%), gentamycin (31.4%), kanamycin (28.6%), erythromycin (25.7%), chloramphenicol (20%), and trimethoprim (14.3%). Meanwhile, only ciprofloxacin, nalidixic acid, and imipenem were indicated as susceptible. Conclusion: The presence of the aerolysin gene is vital in determining the virulence of A. hydrophila. The study results indicated a high aerolysin gene prevalence. In addition, this study emphasized antibiotic use monitoring, food safety improvement, and negative impact reduction on human health and the environment.

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid-ß Peptides with a Nanopore.

Accurate discrimination of amyloid-ß (Aß) peptides containing familial point mutations would advance the knowledge of their roles in early-onset Alzheimer's disease. Herein, we simultaneously identified the mutant A21G, E22G, E22Q, and the wild-type (WT) Aß18-26 peptides with aerolysin nanopore using a 3D blockage mapping strategy. The standard deviation of current blockade fluctuations (σb ) was proposed as a new supplement to current blockage (Ib /I0 ) and duration time (tD ) to profile the blockage characteristics of single molecules. Although the WT and A21G Aß18-26 are indistinguishable in a traditional Ib /I0 -tD 2D description, ∼87 % of the blockade events can be accurately classified with half reduction of false identification using a combination of Ib /I0 , tD, and σb . This work offers an easy and reliable strategy to promote nanopore sensitivity of peptide mutants, leading to a more precise analysis of pathogenic mutations for developing effective diagnosis and treatment.

Inhibitory Effect of Polydatin Against Aeromonas hydrophila Infections by Reducing Aerolysin Production.

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.

On possible trypsin-induced biases in peptides analysis with aerolysin nanopore.

Nanopore-based single-molecule analysis technique is a promising approach in the field of proteomics. In this Technical Brief, the interaction between the biological nanopore of Aerolysin (AeL) and peptides is investigated, focusing on potential biases depending on the AeL activation protocol. Our results reveal that residual trypsin, which may be unintentionally introduced in analyte solution when using a classical AeL activation protocol, can induce a significant formation of shorter peptides by enzymatic degradation of longer ones, which may lead to unwanted effects and/or misinterpretations. AeL free-trypsin activation protocol eliminates this bias and appears more appropriate for peptide/proteins analysis, specifically in the perspective of nanopore-based molecular fingerprinting or of low-abundance species characterization.

Palmatine Inhibits the Pathogenicity of Aeromonas hydrophila by Reducing Aerolysin Expression.

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.

Molecular phylogeny, structure modeling and in silico screening of putative inhibitors of aerolysin of Aeromonas hydrophila EUS112.

Aeromonas hydrophila, a Gram-negative bacterium, causes diseases in fish, resulting in excessive loss to the aquaculture industry. Aeromonas is a highly heterogeneous group of bacteria, and the heterogeneity of the genus is attributed to variation and diversity in the virulence factors and toxins among various Aeromonas strains. One of the major toxins aerolysin, secreted by the bacterium, causes hemorrhagic-septicemia and diarrhea and can serve as a drug target. Here, we describe characterization, molecular phylogeny, and homology modeling of the aerolysin of A. hydrophila strain EUS112 (AhEUS112) cloned in our lab. The encoded aerolysin is 485 amino acids long with an N-terminal signal sequence of 23 amino acids. Phylogenetic analysis of the aerolysin of AhEUS112 revealed that it belongs to a diverse group of toxins, showing maximum similarity with aerolysins of other Aeromonas strains followed by Vibrio toxin. The homology model of the mature aerolysin of AhEUS112 was generated using the crystal structure of a mutant aerolysin (PDB#3g4n) as the template, which showed that the encoded aerolysin exists as a channel protein. Validation of the generated model using bioinformatics tool confirmed it to be a good quality model that can be used for drug design. Molecular dock analysis revealed that drugs, aralia-saponin I, cyclamin, ardisiacrispin B, and aralia-saponin II bind to aerolysin with a higher affinity as compared to other drugs and at functionally important amino acids of aerolysin. Hence, these molecules can act as an effective therapeutics for inhibiting the aerolysin pore formation and curtail the severity of Aeromonas infection.Communicated by Ramaswamy H. Sarma.