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Exo- β - N -acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo - β - N -acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo- β - N -GlcNAcase ( Vh GlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both K m and k cat ), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. Vh GlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC 50 value, of 1.2 μM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants ( K d ) obtained from ITC were 0.19 μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (Δ C p ) of −0.34 ± 0.05 kcal·mol −1 ·K −1 indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases. [ABSTRACT FROM AUTHOR]

A global distribution in marine, brackish, and freshwater ecosystems, in combination with high abundances and biomass, make vibrios key players in aquatic environments, as well as important pathogens for humans and marine animals. Incidents of Vibrio-associated diseases (vibriosis) in marine aquaculture are being increasingly reported on a global scale, due to the fast growth of the industry over the past few decades years. The administration of antibiotics has been the most commonly applied therapy used to control vibriosis outbreaks, giving rise to concerns about development and spreading of antibiotic-resistant bacteria in the environment. Hence, the idea of using lytic bacteriophages as therapeutic agents against bacterial diseases has been revived during the last years. Bacteriophage therapy constitutes a promising alternative not only for treatment, but also for prevention of vibriosis in aquaculture. However, several scientific and technological challenges still need further investigation before reliable, reproducible treatments with commercial potential are available for the aquaculture industry. The potential and the challenges of phage-based alternatives to antibiotic treatment of vibriosis are addressed in this review. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis . The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis ; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios. [ABSTRACT FROM AUTHOR]

SFL, a lectin from the marine sponge Stylissa flexibilis was purified by cold ethanol precipitation followed by ion exchange chromatography on DEAE Sepharose column and Sephacryl S-200 gel filtration. SFL is a dimeric glycoprotein of 32 kDa subunits linked by a disulfide bridge with a molecular mass of 64 kDa by SDS-PAGE and 65 kDa by Sephacryl S-200 gel filtration. SFL preferentially agglutinated enzyme treated human A erythrocytes. The activity of lectin was strongly inhibited by monosaccharide d -galactose and glycoproteins asialo-porcine stomach mucin and asialo-fetuin. The lectin was Ca 2 + dependent, stable over a range of pH from 5 to 8, and up to 60 °C for 30 min. The N-terminal amino acid sequence of SFL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with lectins from the marine sponge Spheciospongia vesparia . SFL caused agglutination of Vibrio alginolyticus and V. parahaemolyticus in a dose dependent manner and inhibited the growth rates of the virulent bacterial strains. Growth inhibition of V. alginolyticus and V. parahaemolyticus with SFL was not observed in the presence of d -galactose or asialo-porcine stomach mucin, suggesting that the lectin caused the agglutination through binding to the target receptor(s) on the surface of Vibrios. Thus, the marine sponge S. flexibilis could promise to be a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent. [ABSTRACT FROM AUTHOR]

A global distribution in marine, brackish, and freshwater ecosystems, in combination with high abundances and biomass, make vibrios key players in aquatic environments, as well as important pathogens for humans and marine animals. Incidents of Vibrio-associated diseases (vibriosis) in marine aquaculture are being increasingly reported on a global scale, due to the fast growth of the industry over the past few decades years. The administration of antibiotics has been the most commonly applied therapy used to control vibriosis outbreaks, giving rise to concerns about development and spreading of antibiotic-resistant bacteria in the environment. Hence, the idea of using lytic bacteriophages as therapeutic agents against bacterial diseases has been revived during the last years. Bacteriophage therapy constitutes a promising alternative not only for treatment, but also for prevention of vibriosis in aquaculture. However, several scientific and technological challenges still need further investigation before reliable, reproducible treatments with commercial potential are available for the aquaculture industry. The potential and the challenges of phage-based alternatives to antibiotic treatment of vibriosis are addressed in this review.

Three isolectins from cultivated Eucheuma denticulatum were isolated. They were commonly monomeric proteins of about 28 kDa with a range of averaged molecular weights from 27,834 to 27,868 Da among the isolectins and shared almost the same 20 N-terminal amino acid sequences. Complementary DNA (cDNA) cloning based on the rapid amplification cDNA ends (RACE) methods elucidated the full-length sequence of EDA-2 which encodes 269 amino acids, including initiating methionine, with four tandemly repeated domains of about 67 amino acids. The primary structure of EDA-2 is highly similar to those of the high-mannose N-glycan specific lectins including Oscillatoria agardhii (OAA) and Burkholderia oklahomensis EO147 (BOA) from cyanobacteria, Myxococcus xanthus (MBHA) and Pseudomonas fluorescens Pf0-1 (PFL) from bacteria, and ESA-2 from a macro red alga. The hemagglutination activities were commonly inhibited by the glycoproteins bearing high-mannose N-glycans, but not by monosaccharides examined, including mannose. In a direct binding experiment with pyridylaminated oligosaccharides, an isolectin EDA-2 exclusively bound to high-mannose type N-glycans, but not to other glycans that include complex types and a core pentasaccharide of N-glycans, indicating that it recognized the branched oligomannoside moiety. Its binding activity was subtly different among the oligomannoside structures examined, showing that the lectin has preference affinity for high-mannose type N-glycans with an exposed (α1-3) mannose residue in the D2 arm. Interestingly, EDAs, the mixture of three isolectins inhibited the growth of shrimp pathogenic bacterium, Vibrio alginolyticus, although it did not affect the growth of V. parahaemolyticus and V. harveyi. Growth inhibition of V. alginolyticus with EDAs was not observed in the presence of yeast mannan bearing high-mannose N-glycans, suggesting that EDAs caused the activity through binding to the target receptor(s) on the surface of V. alginolyticus. These results indicate that cultivated carrageenophyte E. denticulatum is a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent. [ABSTRACT FROM AUTHOR]

Abstract: In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. [Copyright &y& Elsevier]

Aims: To assess the diversity of antibiotic-resistant bacteria and their resistance genes in typical maricultural environments. Methods and Results: Multidrug-resistant bacteria and resistance genes from a mariculture farm of China were analysed via cultivation and polymerase chain reaction (PCR) methods. Oxytetracycline (OTC)-resistant bacteria were abundant in both abalone and turbot rearing waters, accounting for 3·7% and 9·9% of the culturable microbes. Multidrug resistance was common, with simultaneous resistance to OTC, chloramphenicol and ampicillin the most common resistance phenotype. 16S rDNA sequence analyses indicate that the typical resistant isolates belonged to marine Vibrio, Pseudoalteromonas or Alteromonas species, with resistance most common in Vibrio splendidus isolates. For OTC resistance, tet(A), tet(B) and tet(M) genes were detected in some multidrug-resistant isolates, with tet(D) being the most common molecular determinant. For chloramphenicol resistance, cat II was common, and floR was also detected, especially in marine Pseudoalteromonas strains. Conclusions: There is the risk of multidrug-resistant bacteria contamination in mariculture environments and marine Vibrio and Pseudoalteromonas species serve as reservoirs of specific antibiotic resistance determinants. Significance and Impact of the Study: This paper and similar findings from Korea and Japan indicate the potential for widespread distribution of antibiotic resistance genes in mariculture environments from the East Asian region of the world. [ABSTRACT FROM AUTHOR]

Abstract: Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. [Copyright &y& Elsevier]

The coastal and marine environments contain a variety of potentially pathogenic bacteria that can cause cutaneous infection. While some of these infections are relatively minor, some may be life threatening, especially in patients with certain risk factors. The most common and serious of these aquatic-acquired infections are discussed, including 1) the marine vibrios, 2) Mycobacterium marinum , 3) erysipeloid and disseminated Erysipelothrix , and 4) Aeromonas hydrophila . Treatment of these is also discussed. [ABSTRACT FROM AUTHOR]