Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors that are directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified which are mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Genomic Clues of a Multidrug-Resistant Bacterium from Cultured Domestic Silkworm (Bombyx mori L.).

Enterobacter sp. strain ASE was isolated from the gut of an infected domestic silkworm (Bombyx mori L.; Lepidoptera: Bombycidae). The whole-genome sequence (WGS) of the multidrug-resistant strain Enterobacter sp. ASE, which may contribute to our understanding of the strain's antibiotic resistance mechanism and virulence properties.

Trh positive strain of Vibrio parahaemolyticus induce immunity by modulating MAPK pathway: A molecular pathogenic insight in immune-related gene regulation.

Vibrio parahaemolyticus is a zoonotic bacterium that causes infections in shellfish, fish and higher vertebrates as well as in humans. The Tdh and Trh positive strains of V. parahaemolyticus are generally considered as major virulent strains. The pathogenic mechanisms of Trh positive strain of V. parahaemolyticus are poorly understood. Therefore, in the present study Indian Major Carp, Labeo rohita was intraperitoneally challenged with a Trh positive strain of V. parahaemolyticus below lethal dose 50 (LD50) to understand the innate immune response. A significant upregulation in the respiratory burst activity, myeloperoxidase activity and lysozyme activity of serum was observed in the challenged fishes. However, the serum alpha (α) 2-macro globulin activity and antiprotease activity remained unaltered in the infected fish. The relative expression study of some immune-related genes showed that after the experimental challenge the expression of immune-related genes viz., Toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD), Interleukin-1ß (IL-ß), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), Inducible nitric oxide synthase (iNOS), Complement factor 3a (C3a) and Heat shock proteins 70 (Hsp70) was upregulated during infection. Furthermore, overexpression of nuclear factor kappa light chain enhancer of activated B cells (NF-κß), Myeloid differentiation primary response 88 (MyD88), Mitogen-activated protein kinases (MAPK) and cysteine-aspartic proteases (Casp 1) was also observed after post-infection which clearly indicated that Trh positive V. parahaemolyticus activates MAPK pathway. The present study strengthens the understanding of molecular pathogenesis and provides insights on gene regulation during infection with Trh positive V. parahaemolyticus.

Computational characterization and molecular dynamics simulation of the thermostable direct hemolysin-related hemolysin (TRH) amplified from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a major seafood-borne pathogen that causes life-threatening gastroenteric diseases in humans through the consumption of contaminated seafoods. V. parahaemolyticus produces different kinds of toxins, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and some effector proteins belonging to the Type 3 Secretion System, out of which TDH and TRH are considered to be the major factors for virulence. Although TRH is one of the major virulent proteins, there is a dearth of understanding about the structural and functional properties of this protein. This study therefore aimed to amplify the full length trh gene from V. parahaemolyticus and perform sequence-based analyses, followed by structural and functional analyses of the TRH protein using different bioinformatics tools. The TRH protein shares significant conservedness with the TDH protein. A multiple sequence alignment of TRH proteins from Vibrio and non-Vibrio species revealed that the TRH protein is highly conserved throughout evolution. The three dimensional (3D) structure of the TRH protein was constructed by comparative modelling and the quality of the predicted model was verified. Molecular dynamics simulations were performed to understand the dynamics, residual fluctuations, and the compactness of the protein. The structure of TRH was found to contain 19 pockets, of which one (pocket ID: 2) was predicted to be important from the view of drug design. Eleven residues (E138, Y140, C151, F158, C161, K162, S163, and Q164), which are reported to actively participate in the formation of the tetrameric structure, were present in this pocket. This study extends our understanding of the structural and functional dynamics of the TRH protein and as well as provides new insights for the treatment and prevention of V. parahaemolyticus infections.

Appraisal of antioxidant, anti-hemolytic and DNA shielding potentialities of chitosaccharides produced innovatively from shrimp shell by sequential treatment with immobilized enzymes.

Chitosaccharides (CS) of varied size were prepared from shrimp shell through sequential catalysis, using crude protease and chitinase enzymes immobilized on agar beads. In the optimized state, immobilization yield and activity yield for protease were 84% and 62%, and for chitinase were 75% and 57%, respectively. Immobilized protease and chitinase treatment improved CS yields (101 µg/ml) and retained 63% and 52% of activities after 10 reuses, respectively. Stronger radical-scavenging activity (RSA) of CS against ABTS, DPPH and hydroxyl radical was noted with EC50 values 19.1, 26.4 and 29.6 µg/ml, respectively. Peroxyl and superoxide RSAs of 96.8% and 88.6% were noticed at 70 µg/ml of CS. Singlet oxygen quenching, reducing power and ferrous ion-chelating activities of CS were also pronounced. CS reasonably reduced oxidative damage of DNA, protein and RBC by inhibiting H2O2 and AAPH radicals. Reversible CS-DNA condensation leads to DNA stabilization without changing its conformation and advocates its employment in gene therapy.

Biosynthesis, structural architecture and biotechnological potential of bacterial tannase: a molecular advancement.

Tannin-rich materials are abundantly generated as wastes from several agroindustrial activities. Therefore, tannase is an interesting hydrolase, for bioconversion of tannin-rich materials into value added products by catalyzing the hydrolysis of ester and depside bonds and unlocked a new prospect in different industrial sectors like food, beverages, pharmaceuticals, etc. Microorganisms, particularly bacteria are one of the major sources of tannase. In the last decade, cloning and heterologous expression of novel tannase genes and structural study has gained momentum. In this article, we have emphasized critically on bacterial tannase that have gained worldwide research interest for their diverse properties. The present paper delineate the developments that have taken place in understanding the role of tannase action, microbial sources, various cultivation aspects, downstream processing, salient biochemical properties, structure and active sites, immobilization, efforts in cloning and overexpression and with special emphasis on recent molecular and biotechnological achievements.

Characterizing novel thermophilic amylase producing bacteria from taptapani hot spring, odisha, India.

BACKGROUND: Amylases play a vital role in biotechnological studies and rank an important position in the world enzyme market (25% to 33%). Bioprocess method of amylase production is more effective than the other sources, since the technique is easy, cost effective, fast, and the enzymes of required properties can be procured. OBJECTIVES: The current study aimed to report the characteristics of novel amylase producing bacterial strains isolated from Taptapani hot spring, Odisha, India. MATERIALS AND METHODS: Bacterial strains were isolated by dilution plating method from the water samples collected from Taptapani Hot Spring, Odisha and screened for amylase production through starch hydrolysis. The bacterial isolates were identified morphologically, biochemically, and finally by 16S rDNA profiling. RESULTS: Based on the morphological, physiological, biochemical characteristics and the molecular characterization, the isolates SS1, SS2, and SS3 were identified as Bacillus barbaricus, Aeromonas veroni, and Stenotrophomonas maltophilia, respectively. The approximate molecular weight of enzymes from SS1, SS2, and SS3 strains were 19 kDa, 56 kDa and 49 kDa, respectively. CONCLUSIONS: The current report isolates, characterizes, and demonstrates the novel heat-adapted amylase-producing bacteria SS1, SS2 and SS3 from Taptapani hot spring, indicating its potentiality and stability under acidic conditions.

Study on regulation of growth and biosynthesis of cellulolytic enzymes from newly isolated Aspergillus fumigatus ABK9.

This study was aimed to evaluate the pattern of cellulase biosynthesis from Aspergillusfumigatus ABK9 under submerged fermentation. Production was increased concomitantly with fungal growth up to 72 h and reached maximum (Xmax -6.72 g/l) with specific growth rate (mu max) of 0.126/h. Highest specific rate of enzyme production (q ) was found at initial medium pH of 5.0 and incubation temperature of 30 degrees C. At the same time, in the presence of 2-deoxy-D-glucose concentration of 0.5 mg/ml, the production of cellulolytic enzymes, viz, carboxymethyl cellulase activity (CMCase), filter paper degrading activity (FPase) and P-glucosidase activity reached maximum of 132.2, 21.3 and 28.9 U/ml, respectively. Cellulase biosynthesis was induced in respect to higher volumetric production rate (Qp), specific rate of enzymes production (qp, U/g biomass/h) and enzyme/biomass yield (YE/X) when grown in carboxymethyl cellulose in comparison to other saccharides as sole carbon source. Induction ratios (IR) of cellulases were between 12.3 and 24.4 in the presence of 1.5% (w/v) CMC in the culture media. The strain was quite resistant to catabolic repression by glucose up to 0.4% (w/v). Cellulases production was greatly influenced in the presence of yeast extract and potassium dihydrogen phosphate (KH2POA) as nitrogen and phosphate sources in the culture media. C/N ratio of 10.0 and C/P ratio of 4.0 proved to be the best for the production of enzyme cocktail. Along with the high production yield, the crude enzymes showed a promising cellulose hydrolyzing efficiency of rice straw, indicating the enzyme could be beneficial for its large scale industrial exploitation.

Exploitation of fermented shrimp-shells hydrolysate as functional food: assessment of antioxidant, hypocholesterolemic and prebiotic activities.

In the present study the bioactivities of chitooligosaccharides of fermented shrimp-shell hydrolysate (SSH) in respect to hypocholesterolemic, antioxidant and prebiotic activity were tested in male albino rat. Rats were treated with four different diets, viz., (i) cholesterol-rich (5%) basal diet (ChB), (ii) ChB+10% chitin, (iii) ChB+10% SSH and (iv) control group (without cholesterol). After 4 weeks of treatment, body mass index, liver weight, serum total cholesterol and LDL-cholesterol in groups (ii) and (iii) were decreased significantly than group (i). SSH supplementation significantly resists oxidative stress by reducing the thiobarbituric acid reactive substances and by increasing catalase, superoxide dismutase and free radical scavenging activity. The colonization of Lactobacillus and Bifidobacterium population in small and large intestine were more in group (iii) than other groups. Reduction of Clostridium perfringens population and non-significant changes of E. coli was also noted in SSH supplement group. Histological study revealed that the villus height and villus:crypt of the small intestine were increased significantly in SSH supplemented group (iii) without any diarrheal symptoms. The results demonstrated that the shrimp-shells hydrolysate has hypocholesterolemic effect, can resist lipid peroxidation and can influence the growth of health beneficial microbes, hence can be used as functional food for hypercholesterolemic patients.

Analysis of alteration of gut microbial population under the exposure of graded hyperbaric pressures: application of metagenomic approach.

Gastroenterological disorders are very common at hyperbaric conditions. The present study was conducted to find out the impact of gut flora on the gastrointestinal disorders created at such environmental circumstances. For this, male albino rat were exposed to graded hyperbaric pressures (915 and 1277 mmHg) and large intestinal content was examined for microbial composition using culture based and PCR-DGGE tools. After 30 day exposure, total aerobes (38.54 and 375.57 folds, 1.35 and 1.58 gdi) and E. coli (126.05 and 873.23 folds, 1.31 and 1.44 gdi) were increased whereas total anaerobes (7.01 x 10(4) and 8.84 x 10(3) folds, -1.56 and -1.39 gdi), Enterobacter spp. (-2.45 and -1.00 gdi) and Clostridium perfringens (12.88 and 54.16 folds, -1.38 and -1.75 gdi) were decreased significantly in respect to control after exposure of simulated hyperbaric pressures like at 915 and 1277 mmHg, respectively. Metagenomics study revealed an overall reduction in total microbial profile was noted than control at higher level hyperbaric pressure, i.e., 1277 mmHg air pressure for highest duration of exposure. Though, some new bands also appeared which indicated the expansion of dormant or new microbiota, Variation in the numbers of these newly dominated bacteria was correlated to dose and duration of hyperbaric treatment. The histological results clearly indicated that hyperbaric environment induced severe inflammation in the mucosal and submucosal layer of large intestine. Thus, the result suggest that hyperbaric pressure is an important exogenous factor that strongly modulated the intestinal morphology and microbial ecology, and induced several gastrointestinal ailments during hyperbarism.

Collagen scaffolds derived from fresh water fish origin and their biocompatibility.

Collagen, a major component of native extracellular matrix, has diverse biomedical applications. However, its application is limited due to lack of cost-effective production and risk of disease transmission from bovine sources currently utilized. This study describes fabrication and characterization of nano/micro fibrous scaffolds utilizing collagen extracted from fresh water fish origin. This is the first time collagen extracted from fresh water fish origin was studied for their biocompatibility and immunogenicity. The nano/micro fibrous collagen scaffolds were fabricated through self-assembly owing to its amphiphilic nature and were subsequently cross-linked. In vitro degradation study revealed higher stability of the cross-linked scaffolds with only ~50% reduction of mass in 30 days, while the uncross-linked one degraded completely in 4 days. Further, minimal inflammatory response was observed when collagen solution was injected in mice with or without adjuvant, without significant dilution of sera. The fish collagen scaffolds exhibited considerable cell viability and were comparable with that of bovine collagen. SEM and fluorescence microscopic analysis revealed significant proliferation rate of cells on the scaffolds and within 5 days the cells were fully confluent. These findings indicated that fish collagen scaffolds derived from fresh water origin were highly biocompatible in nature.

Regulation of xylanase biosynthesis in Bacillus cereus BSA1.

Microbial xylanases have a promising biotechnological potential to be used in industries. In this study, regulation of xylanase production was examined in Bacillus cereus BSA1. Xylanase production was induced by xylan. The enzyme production further increased in the presence of xylose and arabinose in very low concentration with addition of xylan (0.5% up to 6.02 U/ml). Addition of glucose (about 0.1%) to the media supplemented with xylan repressed xylanase production. Even higher concentration (>0.1%) of xylose and arabinose repressed xylanase biosynthesis. Glucose-mediated repression was partially relived by addition of cyclic adenosine monophosphate. Chemical like 2-4-dinitrophenol, which can inhibit adenosine triphosphate synthesis in cell, repressed xylanase synthesis and it suggested xylanase synthesis to be an energy dependent process.