Enhanced immune defences in Pacific white shrimp (Litopenaeus vannamei) post-exposure to a vibrio vaccine

This study was conducted to determine if exposure of shrimp, Litopenaeus vannamei, to a commercial anti-vibrio vaccine caused changes in antibacterial and cellular (phagocytosis) defences. Shrimp post-larvae were administered either Vibromax™ vaccine or a blank preparation. Whole body homogenates were prepared before (day 0), during (day 10) and after (day 20) vaccination and incubated with a selection of pathogenic vibrios. Homogenate from day 0 animals showed natural antibacterial activity towards Vibrioanguillarum which was significantly enhanced for bacteria-exposed shrimp at 10 days post-challenge. This effect of the vaccine was short-term in its duration. No antibacterial activity was observed in day 0 shrimp homogenate against Vibrio alginolyticus but it was significantly enhanced for both vaccinated and blank-vaccinated shrimp by day 10. No natural or inducible antibacterial activity was observed against Vibrio harveyi at 0, 10 or 20 days post-challenge. To determine if prior exposure of shrimp to inactivated vibrios results in elevated hemocyte phagocytic activity, juveniles were injected with either a mixture of formalin-inactivated vibrios or saline. Hemocyte monolayers made from these shrimp were overlaid with a 1:1 mix of Bacillus subtilis and these vibrios. Hemocytes from vibrio-exposed animals showed elevated levels of internalised vibrios compared with those from the saline injected group. These studies show selectively enhanced cellular defences of shrimp following ‘vaccination’.     

In vitro effects on bacterial growth of phenoloxidase reaction products

An active phenoloxidase preparation from the freshwater crayfish Pacifastacus leniusculus exhibited a strong antibacterial effect in vitro on the bacteria Aeromonas hydrophila, Escherichia coli, Streptococcus pneumoniae whereas a weaker but still significant effect against Bacillus cereus, Pseudomonas aeruginosa and Staphylococcus aureus. In most cases reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa. The effect on bacteria was abolished if no substrate was available for the phenoloxidase or in the presence of the phenoloxidase inhibitor phenylthiourea.     

The anti-platelet drug ticlopidine inhibits FapC fibrillation and biofilm production: Highlighting its antibiotic activity

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.     

Immunosuppressive effect of cyclosporin A on insect humoral immune response

Cyclosporin A suppressed humoral immune response of Galleria mellonella larvae. Insects were immunized with LPS Pseudomonas aeruginosa and then injected with cyclosporin A. Immunosuppressive effects were expressed both, in larvae treated with cyclosporin A at the initial phase of immune response and at the effector phase of antibacterial immunity. Cyclosporin A moderately decreased lysozyme activity and significantly decreased antibacterial activity peptides against Escherichia coli. Immunosuppressive effects of cyclosporin A were observed after immunoblotting with antibodies anti-G. mellonella lysozyme. Tricine SDS/PAGE shown that synthesis of antibacterial peptides of larvae treated with cyclosporin A was considerably inhibited. Insects of impaired immune response by cyclosporin A action lost protective immunity to insect bacterial pathogen P. aeruginosa.     

Insect immunity: Inducible antibacterial activity in Drosophila

Adult Drosophila melanogaster were found to produce an inducible antibacterial activity as a response to an injection (vaccination) of the non-pathogenic bacterium, Enterobacter cloacae. Vaccinated flies showed an increased survival time after a second injection with an insect-pathogenic bacterium, Pseudomonas aeruginosa. This immune response was blocked by cycloheximide, an inhibitor of eukaryotic protein synthesis, a fact which indicates de novo synthesis of the antibacterial factor operating in vivo. Electrophoretic mobility at pH 4 in combination with antibacterial assay and immunological cross reactivity was used to demonstrate attacin-like factors in hemolymph and cecropin-like factors in cell-free extracts of whole flies. Extract of flies also contained lysozyme but this enzyme could not be induced. Some active material is pre-existing and can be released by treatment of whole flies with ultrasound (sonication) or microwaves. Compared to wild-type flies, a mutant strain deficient in leucine aminopeptidase showed much higher values for antibacterial activity and lysozyme.     

Antibacterial activity of quinoxalines,quinazolines, and 1,5-naphthyridines

Several phenyl substituted naphthalenes and isoquinolines have been identified as antibacterial agents that inhibit FtsZ-Zing formation. In the present study we evaluated the antibacterial of several phenyl substituted quinoxalines, quinazolines and 1,5-naphthyridines against methicillin-sensitive and methicillin-resistant Staphylococcus aureus and vancomycin-sensitive and vancomycin-resistant Enterococcus faecalis. Some of the more active compounds against S. aureus were evaluated for their effect on FtsZ protein polymerization. Further studies were also performed to assess their relative bactericidal and bacteriostatic activities. The notable differences observed between nonquaternized and quaternized quinoxaline derivatives suggest that differing mechanisms of action are associated with their antibacterial properties.     

Rational Design of Berberine-Based FtsZ Inhibitors with Broad-Spectrum Antibacterial Activity

Inhibition of the functional activity of Filamenting temperature-sensitive mutant Z (FtsZ) protein, an essential and highly conserved bacterial cytokinesis protein, is a promising approach for the development of a new class of antibacterial agents. Berberine, a benzylisoquinoline alkaloid widely used in traditional Chinese and native American medicines for its antimicrobial properties, has been recently reported to inhibit FtsZ. Using a combination of in silico structure-based design and in vitro biological assays, 9-phenoxyalkyl berberine derivatives were identified as potent FtsZ inhibitors. Compared to the parent compound berberine, the derivatives showed a significant enhancement of antibacterial activity against clinically relevant bacteria, and an improved potency against the GTPase activity and polymerization of FtsZ. The most potent compound 2 strongly inhibited the proliferation of Gram-positive bacteria, including methicillin-resistant S. aureus and vancomycin-resistant E. faecium, with MIC values between 2 and 4 µg/mL, and was active against the Gram-negative E. coli and K. pneumoniae, with MIC values of 32 and 64 µg/mL respectively. The compound perturbed the formation of cytokinetic Z-ring in E. coli. Also, the compound interfered with in vitro polymerization of S. aureus FtsZ. Taken together, the chemical modification of berberine with 9-phenoxyalkyl substituent groups greatly improved the antibacterial activity via targeting FtsZ.     

Smt3 is required for the immune response of silkworm, Bombyx mori

SUMO works in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. The homologous gene of SUMO named BmSmt3 was identified for the first time in silkworm. The expression of BmSmt3 was enhanced in the fat body of silkworm after immune challenge. However, the expression of BmSmt3 after immune challenge was almost invariant in silk gland, which is the nonimmune organ in silkworm. In addition, the expression of BmRelA and CecropinB1 was decreased significantly in pupae after the BmSmt3 was knocked down in vivo. According to our results, BmSmt3 might participate in the immune response through regulating the expression of BmRelA gene, which can further regulate the expression of antibacterial peptide subsequently in silkworm.     

Antibacterial evaluation of silver nanoparticles synthesized from lychee peel: individual versus antibiotic conjugated effects

This paper describes the extracellular synthesis of silver nanoparticles from waste part of lychee fruit (peel) and their conjugation with selected antibiotics (amoxicillin, cefixim, and streptomycin). FTIR studies revealed the reduction of metallic silver and stabilization of silver nanoparticles and their conjugates due to the presence of CO (carboxyl), OH (hydroxyl) and CH (alkanes) groups. The size of conjugated nanoparticles varied ranging from 3 to 10 nm as shown by XRD. TEM image revealed the spherical shape of biosynthesized silver nanoparticles. Conjugates of amoxicillin and cefixim showed highest antibacterial activity (147.43 and 107.95%, respectively) against Gram-negative bacteria i.e. Alcaligenes faecalis in comparison with their control counterparts. The highest reduction in MIC was noted against Gram-positive strains i.e. Enterococcus faecium (75%) and Microbacterium oxydans (75%) for amoxicillin conjugates. Anova two factor followed by two-tailed t test showed non-significant results both in case of cell leakage and protein estimation between nanoparticles and conjugates of amoxicillin, cefixime and streptomycin. In case of MDA release, non-significant difference among the test samples against the selected strains. Our study found green-synthesized silver nanoparticles as effective antibacterial bullet against both Gram positive and Gram negative bacteria, but they showed a more promising effect on conjugation with selected antibiotics against Gram negative type.     

In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.     

Blepharismin produced by a protozoan Blepharisma functions as an antibiotic effective against methicillin-resistant Staphylococcus aureus

A ciliated protozoan, Blepharisma japonicum, produces a photosensitive red pigment, blepharismin (BLR). This study showed that the pigment inhibits the growth of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) resistant to arbekacin (ABK), which is the most effective aminoglycoside antibiotic against MRSA and used world wide. Although the minimum inhibitory concentration (MIC) of BLR to the ABK-resistant MRSA strain was 6.25 μg/ml in dark, it was decreased to 1.25 μg/ml by irradiation with white light of 65 W/m² for 30 min, suggesting that the antibacterial activity of BLR is photoactivated. Our findings suggested that the antibacterial activity of BLR in dark is due to inhibition of protein synthesis. In addition, we found that BLR is bactericidal and enhances synergistically the antibacterial activity of ABK.     

The simultaneous production of single-cell protein and a recombinant antibacterial peptide by expression of an antibacterial peptide gene in Yarrowia lipolytica

In this study, the DNA fragment encoding the N-terminus of scallop H2A was expressed in the marine-derived yeast Yarrowia lipolytica, which has a high protein content. After cultivation in PBB medium for 120 h, the transformant producing the highest amount of antibacterial peptide, 29a, was obtained. The supernatant from cultures of 29a had killing activity against Vibrio harveyi, V. anguillarum, and V. parahaemolyticus. After purification, the molecular mass of the recombinant antibacterial peptide was 4.5 kDa, and the purified recombinant antibacterial peptide was able to cause leakage of intracellular components from both whole and protoplast cells of V. parahaemolyticus. The results indicated that when the yeast transformant 29a was grown in YPD medium, PBB medium or hydrolysate of soybean meal containing ammonium sulfate, its cells still had a high protein content. Because this recombinant marine yeast both had a high protein content and produced the antibacterial peptide, it has high value-added applications.     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

Purification of an antibacterial protein in the coelomic fluid of Nereis diversicolor (annelida,polychaeta). similitude with a cadmium-binding protein

1. Coelomic fluid of Nereis diversicolor shows antibacterial activity 24 hr after inoculation with Escherichia coll, while fluid from untreated control does not.2. This activity is detected by growth inhibition of E. coli or Micrococcus kristinae.3. The thermolabile protein has been purified by two distinct methods using gel filtration and exchange ion chromatography. By SDS-PAGE, the molecular weight of this protein has been estimated at about 10 kDa.4. The protein shows strong similitude with cadmium-binding protein (CBP) which is a dimer constituted by two 10 kDa subunits (Nejmeddine et al., 1988).5. In Western Blot, monoclonal antibody against CBP reacted with the antibacterial protein.6. Moreover, bacteriostatic activity of the coelomic fluid disappeared after incubation with polyclonal and monoclonal antibodies.     

The female reproductive accessory glands of the medfly Ceratitis capitata: Antibacterial activity of the secretion fluid

Secretion from female reproductive accessory glands of the dipteran Ceratitis capitata was found to have antibacterial properties against E. coli. At least two basic polypeptides with mol. wt 15.5 and 4.7 kDa respectively, were identified as responsible for such activity. Furthermore, the 15.5 kDa protein is active against a number of Gram-positive and -negative bacterial strains. Lysozyme activity is also present in the secretion.