Quorum-quenching activity of the AHL-lactonase from <i>Bacillus licheniformis</i> DAHB1 inhibits vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture

Conformation of phylogenetic relationship of penaeidae shrimp based on Morphometric and Molecular investigations

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly Amplified Polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric analysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair-Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable.Понимание точных филогенетических отношений у креветок Penaeidae важно как с общенаучной точки зрения, так и для рыбной промышленности. RAPD анализ был использован для установления филогенетических связей 13 видов креветок Penaeidae. Для морфометрических анализов измерены 40 переменных и общих длин креветок для каждого вида и устранен эффект вариабельности размера. Показатели нормализованного размера обработаны с помощью кластерного анализа UPGMA (Unweighted Pair-Group Method with Arithmetic Mean). При RAPD анализе четыре праймера показали достоверные различия между видами. Коэффициенты корреляции между паттернами ДНК использованы для построения UPGMA дендрограмм. Филогенетические связи, построенные на основе морфометрических и молекулярных анализов, совпали, что позволило сделать вывод об их достоверности

In silico analysis of lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene from the haemocytes of Indian white shrimp Fenneropenaeus indicus

Lipopolysaccharide and β-1,3-glucan binding protein (LGBP) gene are involved in the pattern recognition mechanism of invertebrates, it induces the cell and humoral mediated immune responses like encapsulation, phagocytosis, nodule formation, clotting, synthesis of antimicrobial peptides and activation of the prophenoloxidase (proPO) system. The current study focuses to model the three-dimensional structure of novel immune related gene LGBP from the Indian white shrimp Fenneropeneaus indicus (F.indicus) by in silico homology modeling and its motif prediction. Fenneropeneaus indicus lipopolysaccharide and β-1,3-glucan binding protein (Fein-LGBP) consists of glycosylated regions which come under the glucanase family. Two conserved putative integrin-binding motif (cell adhesion sites), bacterial glucanase motif (GM) and two polysaccharide recognition motifs for the polysaccharide binding motif (PsBM) and β- glucan recognition motif (β-GRM) were conserved in the novel sequences of Fein-LGBP. Prediction of motifs, patterns, disulfide bridges and secondary structure were performed for functional characterization of the Fein-LGBP. Three dimensional structure of the Fein-LGBP was generated by Modeller9V8, Swiss Model and validated using NIH server. Results revealed that the modelled structure of Fein-LGBP was 75.7% of residues in allowed region. Theoretical model of Fein- LGBP facilitates to the discovery of new synthetic immune related peptides, agonists that could be useful to  understand the mechanism of LGBP involvement in the prophenoloxidase activating system of crustaceans. The tertiary structure prediction of the immune related gene Fein- LGBP will assist to explore more knowledge in immune system of crustaceans

Conformation of Phylogenetic Relationship of Penaeidae Shrimp Based on Morphometric and Molecular Investigations

Understanding of accurate phylogenetic relationship among Penaeidae shrimp is important for academic and fisheries industry. The Morphometric and Randomly Amplified Polymorphic DNA (RAPD) analysis was used to make the phylogenetic relationsip among 13 Penaeidae shrimp. For morphometric anal� ysis forty variables and total lengths of shrimp were measured for each species, and removed the effect of size variation. The size normalized values obtained was subjected to UPGMA (Unweighted Pair�Group Method with Arithmetic Mean) cluster analysis. For RAPD analysis, the four primers showed reliable differentiation between species, and used correlation coefficient between the DNA banding patterns of 13 Penaeidae species to construct UPGMA dendrogram. Phylogenetic relationship from morphometric and molecular analysis for Penaeidae species found to be congruent. We concluded that as the results from morphometry investigations concur with molecular one, phylogenetic relationship obtained for the studied Penaeidae are considered to be reliable

N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tigershrimp,Penaeus semisulcatusby gel fermentation chromatography using blue Sepharose matrix. The twopurified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGEand named asPenaeus semisulcatusprophenoloxidase I (PSproPO I) andPenaeus semisulcatusproph-enoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier TransformInfrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography(HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre againsthuman erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activityagainst yeastSaccharomyces cerevisiaeand encapsulation activity against Sepharose CL 6B beadscompared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I andPSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogenassociated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containingPSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria.The above results concluded that no significant differences were found between the purified PSproPO Iand PSproPO II immune indices and functions. This study might provide a sensitive platform to under-stand more about the critical roles of PSproPO I and PSproPO II in crustacean immune syste

Bio-Fabrication of Human Amniotic Membrane Zinc Oxide Nanoparticles and the Wet/Dry HAM Dressing Membrane for Wound Healing

Publication history: Accepted - 25 June 2021; Published online - 28 July 2021.The preparation of unique wet and dry wound dressing products derived from unprocessed human amniotic membrane (UP-HAM) is described. The UP-HAM was decellularized, and the constituent proteins were cross-linked and stabilized before being trimmed and packed in sterile Nucril-coated laminated aluminium foil pouches with isopropyl alcohol to manufacture processed wet human amniotic membrane (PWHAM). The dry type of PD-HAM was prepared by decellularizing the membrane, UV irradiating it, lyophilizing/freeze-drying it, sterilizing it, and storing it at room temperature. The UP-HAM consists of a translucent yellowish mass of flexible membranes with an average thickness of 42 µm. PW-HAM wound dressings that had been processed, decellularized, and dehydrated had a thinner average thickness of 30 µm and lacked nuclear-cellular structures. Following successful decellularization, discrete bundle of fibrous components in the stromal spongy layers, microvilli and reticular ridges were still evident on the surface of the processed HAM, possibly representing the location of the cells that had been removed by the decellularization process. Both wet and dry HAM wound dressings are durable, portable, have a shelf life of 3–5 years, and are available all year. A slice of HAM dressing costs 1.0 US/cm2 . Automation and large-scale HAM membrane preparation, as well as storage and transportation of the dressings, can all help to establish advanced technologies, improve the efficiency of membrane production, and reduce costs. Successful treatment of wounds to the cornea of the eye was achieved with the application of the HAM wound dressings. The HAM protein analysis revealed 360 µg proteins per gram of tissue, divided into three main fractions with MWs of 100 kDa, 70 kDa, and 14 kDa, as well as seven minor proteins, with the 14 kDa protein displaying antibacterial properties against human pathogenic bacteria. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 July 2021 | Volume 9 | Article 695710 fbioe-09-695710 July 22, 2021 Time: 16:39 # 2 Ramasamy et al. HAMP-ZnO Nanoparticles HAM Wound Dressing Wet and dry wound dressings were produced. HAM proteins were purified and analysed. The zinc oxide nanoparticles (HAMP-ZnO NP) made from HAM proteins were characterised and tested for their antibacterial activity. Wounds to the cornea of the eye healed easily when treated with HAM wound dressings. Fresh human Amniotic membrane, Serological screening, selection of disease-free HAM, reome stromal layer, preparation of HAM. UNPROCESSED HAM Cuboidal epithelial cells, basement membrane, compact layer, stromal and spongy layers containing scatted fibroblast cells are visible in hsitological analysis. The flow chart depicts the methods for processing, and preparation of wet (PWHAM) and dry (PD-HAM) wound healing dressings. HAM proteins, Nanoparticle synthesis (HAMP-ZnO NP) and analysis. Antibacterial analysis show Inhibition of growth and biofilm formation of pathogenic bacteria . Processed HAM lacked a nuclear-cellular epithelium, but it did have a distinct fibrous elements in basement membrane, stromal and spongy layers. Processed PW-HAM (Light &SEM) showed smooth epithelial surface topography with microvilli,. HAM dressing, wet/dry, packed, labelled, sterilised and processed. They are durable, portable, have long shelf life . A slice of HAM dressing costs US 1.0 / cm² . The wound dressings are ready to be applied. The dermal wounds and conjunctival surface can be successfully repaired using processed HAM wound dressings GRAPHICAL ABSTRACT | Flow chart depicting the methods, preparing, and characterizing, by histological, and scanning electron microscopy, of wet (PW-HAM) and dry (PD-HAM)of wound healing dressing, and preparation of nanoparticles (HAMP ZnO NP); and application of HAM wound dressing. A wide range of antibacterial activity was observed after treatment with 75 µg/ml zinc oxide nanoparticles derived from human amniotic membrane proteins (HAMP-ZnO NP), including dose-dependent biofilm inhibition and inhibition of Gram-positive (S. aureus, S. mutans, E. faecalis, and L. fusiformis) and Gram-negative bacteria (S. sonnei, P. aeruginosa, P. vulgaris, and C. freundii).PR has acknowledged Sree Balaji Medical College and Hospital for providing the article processing charges of the journal, and moral and technical support. The support of Cologenesis Health Care Pvt. Ltd. for a study on “Human amniotic membrane for ocular and dermal applications” is sincerely appreciated

A Nonluminescent and Highly Virulent Vibrio harveyi Strain Is Associated with “Bacterial White Tail Disease” of Litopenaeus vannamei Shrimp

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by “white tail” and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of “white tail” but of non-bacterial origin, the present disease was named as “bacterial white tail disease (BWTD)”. Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system

A 3D Model of the Membrane Protein Complex Formed by the White Spot Syndrome Virus Structural Proteins

Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process.In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers.From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented