Gram-negative Bacterium Aeromonas Hydrophila Research Articles

Effects of berberine hydrochloride on antioxidant response and gut microflora in the Charybdis japonica infected with Aeromonas hydrophila

This study used berberine hydrochloride to treat the Asian paddle crab, Charybdis japonica infected with the Gram-negative bacterium Aeromonas hydrophila at concentrations of 0, 100, 200 and 300 mg/L. The effect of berberine hydrochloride on the survival rate and gut microbiota of C. japonica was investigated. Berberine hydrochloride improved the stability of the intestinal flora, with an increase in the abundance of probiotic species and a decrease in the abundance of both pathogenic bacteria after treatment with high concentrations of berberine hydrochloride. Berberine hydrochloride altered peroxidase activity (POD), malondialdehyde (MDA), and lipid peroxidation (LPO) in the intestinal tract compared to the control. Berberine hydrochloride could modulate the energy released from the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) in the intestinal tract of C. japonica infected with A. hydrophila. Zona occludens 1 (ZO-1), Zinc finger E-box binding homeobox 1 (ZEB1), occludin and signal transducer, and activator of transcription5b (STAT5b) expression were also increased, which improved intestinal barrier function. The results of this study provide new insights into the role of berberine hydrochloride in intestinal immune mechanisms and oxidative stress in crustaceans.

Deciphering genome-wide molecular pathways for exogenous Aeromonas hydrophila infection in wide-bodied sand loach (Sinibotia reevesae)

Aquaculture, as a rapidly growing industry, plays a significant role in global food production and economic development. However, various infectious diseases, particularly those caused by the gram-negative bacterium Aeromonas hydrophila, pose a critical threat to the sustainability and productivity of world-wide aquaculture operations. Investigating the molecular mechanisms of A. hydrophila infection can help to identify key targets for therapeutic interventions and vaccines, thereby reducing economic losses associated with disease outbreaks. Our present study focuses on wide-bodied sand loach (WBSL, Sinibotia reevesae), a captive-bred commercial fish, to explore detailed molecular effects of exogenous A. hydrophila infection. Firstly, a high-quality chromosome-level haplotype genome with a total length of 774 Mb and 49 chromosomes was assembled, providing valuable insights into its genetic components. Secondly, we isolated and characterized a newfound A. hydrophila F4S strain, examined pathological symptoms of WBSL after infection, and then investigated dynamic gene expressions. Our results revealed that the bacterial infection decreased metabolic activity, causing disruptions in cardiac function, cellular homeostasis, tissue damages, and impaired energy utilization. The immune system responds multifaceted, potentially impacting overall health and survival. In summary, our findings contribute to the understanding of the complicated interactions between WBSL and exogenous A. hydrophila, especially shed lights on the genetic complexity, immune responses, and potential impacts on fish health and survival. Furthermore, this study provides new insights into the mechanisms underlying A. hydrophila infection, and offers potential strategies to offload corresponding pathological effects, not only in WBSL but also in other aquaculture species.

Expression profiling and antibacterial analysis of cd36 in mandarin fish, Siniperca chuatsi.

Cd36 is classified as a class B scavenger receptor and has also been identified as a pattern recognition receptor. In this study, we investigated the genomic structure and molecular characteristics of cd36 in mandarin fish (Siniperca chuatsi), examined its tissue distribution, and evaluated its antibacterial activity. Genomic structure analysis showed that Sccd36 consists of 12 exons and 11 introns. Sequencing analysis confirmed that the open reading frame of Sccd36 contains 1410 bp, encoding 469 amino acids. Sccd36 is deeply conserved with other vertebrates in terms of genomic structure, gene loci and molecular evolution, and the feature of two transmembrane was observed in ScCd36 through structural prediction. Sccd36 was constitutively expressed in all tissues tested, with the strongest expression in the intestine, followed by the heart and the kidney. Dramatic changes of Sccd36 mRNA were detected in mucosal tissues, including the intestine, gill and skin, when stimulated by the microbial ligands lipopolysaccharide and lipoteichoic acid. In addition, ScCd36 was identified as having strong binding ability to microbial ligands and antibacterial activity against the gram-negative bacteria Aeromonas hydrophila and the gram-positive bacteria Streptococcus lactis. Furthermore, we verified that the genetic ablation of cd36 impaired the resistance of fish to bacterial challenge by using zebrafish cd36 knockout line. In conclusion, our findings suggest that ScCd36 plays a crucial role in the innate immune response of mandarin fish against bacterial infections. This also sets the stage for further exploration into the antibacterial function of Cd36 in lower vertebrate species.

Study on Phytochemical Profiling of Euphorbia cyathophora Leaf Extracts and Their Potential Antimicrobial, Larvicidal, and Pupicidal Properties Against Earias vittella

Earias vittella is a serious polyphagous agricultural pest that can directly or indirectly damage crops and stored products worldwide. Using synthetic chemicals in pest control has resulted in physiological resistance and adverse environmental effects. However, this alternative to plant-based pest control systems is ecologically friendly and biodegradable. Moreover, Euphorbia cyathophora is a medicinal plant used for many therapeutic activities. Therefore, the present study is aimed at E. cyathophora leaf extract evaluation of the phytochemical, in vitro antimicrobial, larvicidal, and pupal activity of E. vittella. A preliminary phytochemical screening of E. cyathophora leaf extracts revealed numerous bioactive components such as alkaloids, flavonoids, sterols, terpenoids, anthraquinones, carbohydrates, and essential oils. GC-MS analysis of E. cyathophora leaf different extracts revealed the presence of 29 phytochemicals. Among the extracts tested, the methanolic leaf extract showed the highest range of growth inhibition against gram-positive bacteria Streptococcus iniae (15 ± 0.26) and gram-negative bacteria Aeromonas hydrophila (13 ± 0.6), while antifungal activity against Aspergillus flavus (14 ± 0.2) and Aspergillus niger (12 ± 0.36) compared to other solvent extracts. In addition, the bioefficacy of E.Cyathophora leaf extract treated against IV instar larvae and pupae of E. vittella; however, the highest larval and pupal mortality was observed in the methanol extract at 750 ppm, followed by acetone and chloroform against E. vittella. Larvicidal activity of E. cyathophora leaf extracts showed LC50 and LC90 values: methanol 298.64 ppm (708.14); Acetone 362.61 ppm (848.56), and chloroform 415.18 ppm (937.31), respectively. Similarly, thepupicidal effect of E. Cyathophora leaf extracts showed LC50 and LC90 values: methanol 470.25 ppm (958.01); acetone 527.36 ppm (996.35) and chloroform 545.55 ppm (997.88). This study strongly suggests that E. cyathophora leaf extract exhibited significant larval and pupal mortality and potential antimicrobial activity, which can be observed as the development of a new botanical preparation for economic pest control.

ВЕТЕРИНАРНО-САНИТАРНАЯ ЭКСПЕРТИЗА КАРПА, ВЫРАЩЕННОГО В РЫБОХОЗЯЙСТВЕННОМ ВОДОЕМЕ

Modern forms of pond fish farming provide for compacted planting of fish in ponds. This causes close contact of farmed fish and favorable conditions for the spread of diseases. Most often carp fish get sick. A veterinary and sanitary examination of fish caught from the ponds of the Institute of Fish Farming and Biserovskiy fish plant cages was carried out according to organoleptic and bacteriological indicators. An autopsy of the fish was carried out in order to identify endoparasites, ectoparasites and changes in internal organs. As a result of the veterinary and sanitary examination, inflammation of the swim bladder was noted in some fish on the basis of clinical data and parasitological research. No parasites were found. No significant changes were registered during the organoleptic examination. In sick fish, when cooking, there was a slight turbidity and a decrease in the size of fat droplets on the surface of the broth. Bacterioscopic examination of carp revealed gram-negative bacterium aeromonas hydrophila, which causes aeromonosis. Recommendations for the prevention of inflammation of the swim bladder and aeromonosis are given.

Functional Characterization of Serum Amyloid P Component (SAP) in Host Defense against Bacterial Infection in a Primary Vertebrate.

Serum amyloid P component (SAP), an ancient short pentraxin of the pentraxin family, plays an essential role in resistance to bacterial infection. In this study, the expression and functional characterization of SAP (OnSAP) in Nile tilapia (Oreochromis niloticus), a primary vertebrate, are investigated. The open reading frame of OnSAP is 645 bp of a nucleotide sequence encoding a polypeptide of 214 amino acids. As a calcium-binding protein, the structure and relative motif of OnSAP is highly similar to those of humans, containing amino acid residues Asn, Glu, Gln and Asp. In healthy fish, OnSAP mRNA is extensively distributed in all eleven tissues examined, with the highest level in spleen. The mRNA expression of OnSAP was significantly up-regulated after being challenged with gram-positive bacterium Streptococcus agalactiae and gram-negative bacterium Aeromonas hydrophila in vivo. In addition, recombinant OnSAP ((r)OnSAP) protein had capacities of binding S. agalactiae or A. hydrophila in the presence of Ca2+. Further, (r)OnSAP helped monocytes/macrophages to efficiently phagocytize bacteria. Moreover, the (r)OnSAP was able to enhance the complement-mediated lysis of the chicken red blood cells. Collectively, the evidence of SAP in tilapia, based on the results including its evolutionary conserved protein structure, bacterial binding and agglutination, opsonophagocytosis of macrophage and hemolysis enhancement, enriches a better understanding of the biological functions of the pentraxin family.

Tinospora cordifolia- leaves Aqueous Extract Biocidal Activity on Gram-Negative Bacteria

Tinospora cordifolia- leaves aqueous extract is having strong biocidal properties and used as a main home remedial plant since ancient times. Herbal extraction is having slow-acting properties, so a single dose is not effective as allopathic medicine, perhaps long-term exposure is required. Biocidal activity of leaves aqueous extract is tested using human gastro-intestinal gram-negative pathogenic bacterium Aeromonas hydrophila isolated from the wastewater treatment plant. Satisfactory results were obtained that confirm the safety of Tinospora cordifolia leaves aqueous extraction as an effective home remedial as well as pharmaceutical product due to having effective biocidal properties. To the best of our knowledge, aqueous extraction of Tinospora cordifolia leaves with its biocidal activity is first reported in this study.

Photodynamic Inactivation of Antibiotic-Resistant and Sensitive Aeromonas hydrophila with Peripheral Pd(II)- vs. Zn(II)-Phthalocyanines.

The antimicrobial multidrug resistance (AMR) of pathogenic bacteria towards currently used antibiotics has a remarkable impact on the quality and prolongation of human lives. An effective strategy to fight AMR is the method PhotoDynamic Therapy (PDT). PDT is based on a joint action of a photosensitizer, oxygen, and light within a specific spectrum. This results in the generation of singlet oxygen and other reactive oxygen species that can inactivate the pathogenic cells without further regrowth. This study presents the efficacy of a new Pd(II)- versus Zn(II)-phthalocyanine complexes with peripheral positions of methylpyridiloxy substitution groups (pPdPc and ZnPcMe) towards Gram-negative bacteria Aeromonas hydrophila (A. hydrophila). Zn(II)-phthalocyanine, ZnPcMe was used as a reference compound for in vitro studies, bacause it is well-known with a high photodynamic inactivation ability for different pathogenic microorganisms. The studied new isolates of A. hydrophila were antibiotic-resistant (R) and sensitive (S) strains. The photoinactivation results showed a full effect with 8 µM pPdPc for S strain and with 5 µM ZnPcMe for both R and S strains. Comparison between both new isolates of A. hydrophila (S and R) suggests that the uptakes and more likely photoinactivation efficacy of the applied phthalocyanines are independent of the drug sensitivity of the studied strains.

An Ulcerating Perineal Rash in an 8-month-old Girl.

An Ulcerating Perineal Rash in an 8-month-old Girl.

The N‐ and C‐terminal carbohydrate recognition domains of galectin‐9 from Carassius auratus contribute differently to its immunity functions to Aeromonas hydrophila and Staphylococcus aureus

Galectin-9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen-associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin-9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal-9. The predicted CaGal-9 protein contained two non-identical carbohydrate recognition domains (CRDs), namely, N-CRD and C-CRD. The recombinant proteins (rCaGal-9, rN-CRD and rC-CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D-galactose, α-lactose and N-acetyl-D-galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram-negative bacterium Aeromonas hydrophila and Gram-positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal-9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus.

Bio-directed synthesis of Pt-nanoparticles from aqueous extract of red algae Halymenia dilatata and their biomedical applications

In this study, the biosynthesis of platinum nanoparticles (Hd-PtNPs) from red algae Halymenia dilatata and its broad spectrum of bioactivities were explored. The synthesized Hd-PtNPs were characterized by UV–visible spectroscopy, X-ray diffraction (XRD), fourier-transform infrared spectroscopy (FT-IR), field emission scanning electron microscope (FE-SEM), high-resolution transmission electron microscope (HR-TEM), and dynamic light scattering (DLS) analysis. The XRD results showed that the synthesized particles were pure crystalline while the FE-SEM and HR-TEM analysis demonstrated that the Hd-PtNPs were spherical and well-dispersed with an average size of 15 ± 1.7 nm. The zeta potential of Hd-PtNPs was found to be −19.9 mV, which indicates that the synthesized particles were highly stable and well-dispersed. The presence of phytochemical constituents in the H. dilatata aqueous extract performs a capping and reducing process in the Hd-PtNPs formation, which was confirmed by FT-IR analysis. The synthesized Hd-PtNPs showed excellent antioxidant properties in DPPH radical scavenging, reducing power, and total antioxidant assays. Furthermore, Hd-PtNPs exhibit a strong cytotoxic effect against MDA-MB-231 cancer cell lines (IC50 = 50 μg/mL). Moreover, the Hd-PtNPs showed notable inhibitory activity against Gram-positive (Streptococcus pneumonia) and Gram-negative bacteria (Aeromonas hydrophila). In addition, Hd-PtNPs display less toxicity effect on Artemia nauplii even at a higher concentration corroborating the fact that the synthesized Hd-PtNPs are eco-friendly. Thus, Hd-PtNPs can be used as an alternative source in pharmaceutical and biomedical applications.

Expansion of Necrosis Depending on Hybrid Motor-Driven Motility of Aeromonas hydrophila in a Murine Wound Infection Model

The gram-negative bacterium Aeromonas hydrophila is a cause of fulminant and lethal necrotizing soft tissue infections (NSTIs). Suppressing the rapid proliferation of the pathogen and expansion of the necrosis caused in the host is an important issue in clinical practice, but the pathogenic mechanism for the rapid aggravation has not been clarified. In this study, we characterized the function of two types of motor stators in A. hydrophila and explored the role of motility during wound infection. In vitro analysis showed that the motility was reliably maintained while being complemented by the stators. We created a non-motile strain that lacked genes encoding two types of motor stators and analyzed the role of motility in a murine wound infection model. Examination of the bacterial burden in the local infection site and systemic circulation revealed that motility was not essential for the proliferation of A. hydrophila in the host. However, the extent of necrosis at the lesions was lower, and survival times were prolonged in mice infected with the non-motile strain compared with mice infected with the parent strain. These results provide evidence that the rapid expansion of necrosis and the progression to death within a short time period is dependent on the motility of A. hydrophila.

Cloning and functional characterisation of natural killer enhancing factor-B (NKEF-B) gene of Labeo rohita: Anti-oxidant and antimicrobial activities of its recombinant protein

Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 μM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.

Effect of dietary isoleucine on skin mucus barrier and epithelial physical barrier functions of hybrid bagrid catfish Pelteobagrus vachelli × Leiocassis longirostris.

The study investigated the effects of dietary isoleucine (Ile) on skin mucus barrier and epithelial physical barrier functions of hybrid bagrid catfish Pelteobagrus vachelli × Leiocassis longirostris. A total of 630 fish (33.11 ± 0.09g) were fed semi-purified isonitrogenous diets containing 5.0 (control), 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0g Ile kg -1 diet for 8weeks. The results indicated that dietary Ile increased (P < 0.05) in skin (1) mucus protein content and antimicrobial activity against three gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, and Yersinia ruckeri) and two gram-positive bacteria (Streptococcus agalactiae and Staphylococcus aureus), (2) mucus lysofew information is available about the influencezyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities, and complement 3 and 4 (C3 and C4) and immunoglobulin M (IgM) contents, (3) intelectin 1 (intl1), intelectin 2 (intl2), c-type-lysozyme (c-LZM), g-type-lysozyme (g-LZM), and β-defensin mRNA levels. Dietary Ile decreased (P < 0.05) reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC) contents, and up-regulated (P < 0.05) CuZnSOD, GST, GPX1a, muc5ac, muc5b, zonula occludens-1 (ZO-1), zonula occludens-2 (ZO-2), occludin, and claudin 3 mRNA levels in skin. These results indicated that Ile improved skin mucus barrier function via increasing mucus protein, C3 and C4, and IgM contents and antibacterial factors activities, and promoted epithelial physical barrier function via decreasing skin antioxidant damage and improving tight junction structure in hybrid bagrid catfish.

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

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

Molecular and functional characterisation of a mannose-binding lectin-like gene from Japanese sea bass (Lateolabrax japonicus)

Mannose-binding lectin (MBL) plays an important role in host immune responses against pathogens. LjMBL-like-1 was identified from Japanese sea bass (Lateolabrax japonicas), which has selectivity for galactose. Herein, this lectin might be better designated as galactose-binding lectin (LjGalBL-1). LjGalBL-1 transcripts were detected in all tested tissues, with highest expression in liver. Upon Vibrio harveyi infection, LjGalBL-1 mRNA expression was increased in major immune-related tissues, and protein levels in serum were also upregulated. Recombinant LjGalBL-1 (rLjGalBL-1) bound to monosaccharides and polysaccharides, and both rLjGalBL-1 and native LjGalBL-1 (nLjGalBL-1) agglutinated three Gram-positive bacteria (Staphylococcus aureus, Streptococcus iniae and Micrococcus luteus) and four Gram-negative bacteria (Aeromonas hydrophila, Edwardsiella tarda, Vibrio anguillarum and V. harveyi) in a Ca2+-dependent manner in vitro. Moreover, rLjGalBL-1 increased the survival rate of V. harveyi-infected fish and decreased bacterial load in liver, spleen, kidney and blood. Thus, LjGalBL-1 protects L. japonicas against V. harveyi infection.

A novel globular C1q domain containing protein (C1qDC-7) from Crassostrea gigas acts as pattern recognition receptor with broad recognition spectrum

The globular C1q domain containing (C1qDC) proteins are a family of versatile pattern recognition receptors (PRRs) to bind various ligands by their globular C1q (gC1q) domain. In the present study, a novel globular C1qDC (CgC1qDC-7) was characterized from Pacific oyster Crassostrea gigas. The open reading frame of CgC1qDC-7 was of 555 bp, encoding a polypeptide of 185 amino acids. Phylogenetic analysis indicated that CgC1qDC-7 shared high homology with C1qDCs from Crassostrea virginica, Mytilus galloprovincialis, and Mizuhopecten yessoensis. The mRNA transcripts of CgC1qDC-7 were widely expressed in all the tested tissues including mantle, gonad, gills, adductor muscle, hemocytes, hepatopancreas and labial palps, with the highest expression level in hemocytes and gills. The recombinant protein of CgC1qDC-7 (rCgC1qDC-7) exhibited binding activity towards Gram-negative bacteria (Vibrio splendidus, V. anguillarum, Escherichia coli, V. alginolyticus, and Aeromonas hydrophila), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and fungi (Pichia pastoris and Yarrowia lipolytica), and displayed strongest binding affinity towards Gram-negative bacteria V. splendidus and V. anguillarum. It also exhibited affinity to vital pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C) with high affinity towards LPS and PGN, and low affinity to MAN and Poly (I:C). These results collectively indicated that CgC1qDC-7 was a novel PRR in C. gigas with high binding affinity towards LPS and PGN as well as Gram-negative bacteria.

Identification and characterization of a B-type mannose-binding lectin from Nile tilapia (Oreochromis niloticus) in response to bacterial infection

Lectins are a group of carbohydrate-binding proteins, which play an important role in innate immune system against pathogen infection. In this study, a B-type mannose-binding lectin (OnBML) was identified from Nile tilapia (Oreochromis niloticus), and characterized at expression patterns against bacterial infection and capability to promote phagocytosis by macrophages. The open reading frame of OnBML is 354 bp of nucleotide sequence encoding polypeptides of 117 amino acids. The deduced protein is highly homologous to other teleost BMLs, containing two repeats of the conserved mannose-binding motif QXDXNXVXY. Expression of OnBML was widely exhibited in all examined tissues, with the most abundance in spleen and following gill, peripheral blood, and head kidney. The OnBML expressions were significantly up-regulated following two major bacterial infections including a Gram-positive bacterium (Streptococcus agalactiae) and a Gram-negative bacterium (Aeromonas hydrophila) in vivo and in vitro. Recombinant OnBML protein possessed capacities of mannose-binding and calcium-dependent agglutination to S. agalactiae and A. hydrophila, and promoted the phagocytosis by macrophages. Taken together, the present study indicated that OnBML is likely to get involved in host defense against bacterial infection in Nile tilapia.

Identification and bioactivity analysis of a newly identified defensin from the oyster Magallana gigas

The relatively conserved sequences of signal peptides and proregions that antimicrobial peptides (AMPs) contain have been successfully used to search for and identify novel AMPs from databases within the same lineages of fish and amphibians and across different animal classes. If such an approach is applicable to invertebrate species such as oyster has not yet been tested so far. In this study, we found a cDNA from the digestive gland of the oyster Magallana gigas, designated Mgdefdg, which contains two exons interspaced by one intron. Mgdefdg coded for a protein with features characteristic of defensins. The mature peptide had the cysteine-stabilized α-helix/β-sheet motif (CSαβ) and the consensus pattern C-X5-6-C-X3-C-X4-6-C-X3-4-C-X7-8-C-X-C-X2-C forming potential disulfide linkages C1-C5, C2-C6, C3-C7 and C4-C8 in the predicted tertiary structure. Functional assays revealed that recombinant mature MgDefdg (rmMgDefdg) was able to kill the Gram-negative bacterium Aeromonas hydrophila and the Gram-positive bacterium Staphylococcus aureus, and to induce bacterial membrane/cytoplasmic damage. ELISA showed that rmMgDefdg had high affinity to both A. hydrophila and S. aureus as well as the microbe-associated molecular pattern molecules LPS and LTA. Moreover, rmMgDefdg was capable of causing bacterial membrane permeabilization and depolarization, and intracellular ROS increase. Additionally, rmMgDefdg was not cytotoxic to human red blood cells and murine RAW264.7 cells. Taken together, our results indicate that MgDefdg is a previously uncharacterized defensin with membrane selectivity towards bacterial cells. It also shows that the use of conserved sequences of signal peptides of defensins can be an effective tool to identify potential defensins across different animal genera in invertebrates.

Engineering Pichia pastoris for Efficient Production of a Novel Bifunctional Strongylocentrotus purpuratus Invertebrate-Type Lysozyme.

Lysozymes are known as ubiquitously distributed immune effectors with hydrolytic activity against peptidoglycan, the major bacterial cell wall polymer, to trigger cell lysis. In the present study, the full-length cDNA sequence of a novel sea urchin Strongylocentrotus purpuratus invertebrate-type lysozyme (sp-iLys) was synthesized according to the codon usage bias of Pichia pastoris and was cloned into a constitutive expression plasmid pPIC9K. The resulting plasmid, pPIC9K-sp-iLys, was integrated into the genome of P. pastoris strain GS115. The bioactive recombinant sp-iLys was successfully secreted into the culture broth by positive transformants. The highest lytic activity of 960U/mL of culture supernatant was reached in fed-batch fermentation. Using chitin affinity chromatography and gel-filtration chromatography, recombinant sp-iLys was produced with a yield of 94.5mg/L and purity of > 99%. Recombinant sp-iLys reached its peak lytic activity of 8560U/mg at pH 6.0 and 30°C and showed antimicrobial activities against Gram-negative bacteria (Vibrio vulnificus, Vibrio parahemolyticus, and Aeromonas hydrophila) and Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In addition, recombinant sp-iLys displayed isopeptidase activity which reached the peak at pH 7.5 and 37°C with the presence of 0.05M Na+. In conclusion, this report describes the heterologous expression of recombinant sp-iLys in P. pastoris on a preparative-scale, which possesses lytic activity and isopeptidase activity. This suggests that sp-iLys might play an important role in the innate immunity of S. purpuratus.