Evaluation of Therapeutic Efficiency of Stylicin against Vibrio parahaemolyticus Infection in Shrimp Penaeus vannamei through Comparative Proteomic Approach.

The shrimp immune system defends and protects against infection by its naturally expressing antimicrobial peptides. Stylicin is a proline-rich anionic antimicrobial peptide (AMP) that exhibits potent antimicrobial activity. In this study, stylicin gene was isolated from Penaeus vannamei, cloned into vector pET-28a ( +), and overexpressed in Escherichia coli SHuffle T7 cells. The protein was purified and tested for its antibiofilm activity against shrimp pathogen Vibrio parahaemolyticus. It was resulted that the recombinant stylicin significantly reduced the biofilm formation of V. parahaemolyticus at a minimum inhibitory concentration (MIC) of 200 µg. Cell aggregation was observed by using scanning electron microscopy and confocal laser scanning microscopy, and it was resulted that stylicin administration significantly affects the cell structure and biofilm density of V. parahaemolyticus. In addition, real-time PCR confirmed the downregulation (p < 0.05) of genes responsible for growth and colonization. The efficacy of stylicin was tested by injecting it into shrimp challenged with V. parahaemolyticus and 7 days after infection, stylicin-treated animals recovered and survived better in both treatments (T2-100 µg stylicin, - 68.8%; T1-50 µg stylicin, 60%) than in control (7%) (p < 0.01). Comparative proteomic and mass spectrometry analysis of shrimp hemolymph resulted that the expressed proteins were involved in cell cycle, signal transduction, immune pathways, and stress-related proteins representing infection and recovery, and were significantly different in the stylicin-treated groups. The result of this study suggests that the stylicin can naturally boost immunity and can be used as a choice for treating V. parahaemolyticus infections in shrimp.

Functional and molecular characterization of a cold-active lipase from Psychrobacter celer PU3 with potential antibiofilm property.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129-135 AA) and R2 (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Molecular docking study of bio-inhibitors extracted from marine macro-alga Ulva fasciata against hemolysin protein of luminescence disease-causing Vibrio harveyi.

Shrimp grow-out and hatchery systems are being affected by bacterial disease particularly Vibrios. The use of chemotherapeutic agents in aquaculture practices has to lead to the development of resistance among aquatic bacteria. Thus, health management becomes of major importance in aquaculture. Under this situation, progressing bio-inhibitors from marine resources are most appropriate to be considered against pathogenic bacteria. Molecular docking is an appropriate tool in structural biology and computer-assisted drug design to predict and neutralize a target protein of known diseases. In this study, marine macro-alga Ulva fasciata was aimed at developing inhibitors against luminescence disease-causing pathogenic bacteria Vibrio harveyi. U. fasciata was collected from Thoothukudi, Tamil Nadu, India. Extract of U. fasciata was tested against growth and virulence factors of V. harveyi during Penaeus monodon larviculture. Further U. fasciata extract was subjected to GC-MS analysis to identify the biomolecules. The homology modeling of virulent protein, hemolysin of V. harveyi was designed in this study. Hence, it was aimed for molecular docking against the biomolecules identified from U. fasciata extract. During shrimp larviculture, the extract of U. fasciata (200 µg mL-1) exhibited reduction on Cumulative Percentage of Mortality (32.40%) in postlarvae against challenge of V. harveyi infection. Biomolecule Methyl dehydroabietate had showed highest binding affinity among the compounds was evaluated in molecular docking study. Statistical analysis had revealed significant differences (p < 0.05) in trials. Therefore, it was proved that the bio-inhibitors from U. fasciata will be a better option for controlling luminescence disease-causing V. harveyi in shrimp grow-out practices.

Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.

Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.