-
Journal of Invertebrate Pathology Jun 2022Due to the fast growth rate, the short life cycle, the high market price and the high food conversion efficiency, O. vulgaris is considered as a good candidate for...
In vitro hemocyte phagocytosis activation after experimental infection of common octopus, Octopus vulgaris (Cuvier, 1797) with Photobacterium damselae subsp. piscicida or Vibrio alginolyticus at different temperatures and infection routes.
Due to the fast growth rate, the short life cycle, the high market price and the high food conversion efficiency, O. vulgaris is considered as a good candidate for aquaculture. One of the prerequisites for the successful integration of new species, such as octopi, into industrial-scale production, is the knowledge of the pathological conditions that may arise, with emphasis on infectious diseases caused by microorganisms and para-sites transmitted through wild populations, especially for the farmed organisms cul-tured in cages in proximity to teleost fish. The main objectives of this study were to investigate the sensitivity of common octopus to experimental infection with pathogenic bacteria, to assess the activation of hemocytes and more specifically their phagocytic activity after infection and to associate sensitivity of the species and phagocytic activity of hemocytes to temperature changes, route of infection and pathogen. Common octopus individuals were intramuscularly and intravenously infected with either Photobacterium damselae subsp. piscicida or Vibrio alginolyticus. The hemocyte phagocytosis activation in vitro at two temperatures (21 ± 0.5 °C and 24 ± 0.5 °C) was studied, in an effort to relate these aspects to climate change. Hemolymph was withdrawn on days 0, 3 and 7 post infections/injections. Number of circulating hemocytes/ml hemolymph, phagocytosis ability and Phagocytosis Particle Binding Intensity index were determined. Correlations between hemocytes and bodyweight and between hemocytes and phagocytosis ability were also determined. No mortalities were recorded irrespective of pathogen, route of infection and temperature employed. Circulating hemocytes in control specimens ranged between 1.60x10 hemocytes ml hemolymph to 20.02x10 hemocytes/ml hemolymph at both experiments and temperatures. The interrelation between octopi weight and circulating hemocytes showed that natural fluctuations, age, maturity stage and temperature may affect this relationship. Rise of temperature influenced phagocytosis which seemed to be route of infection, time-point and pathogen related. Specimens infected with Photobacterium damselae subsp. piscicida showed decreased phagocytosis with rise of temperature while when Vibrio alginolyticus was used, phagocytosis activity increased, in most cases. Temperature also played an important role in the correlation between the circulating hemocytes and phagocytosis activity, as at lower temperatures a negative strong correlation was observed. The results prompted us to calculate the activation index. This index showed that temperature is an important factor in hemocyte activation since for Photobacterium damselae subsp. piscicida infected specimens, hemocytes were more activated at 21 ± 0.5℃ instead of 24 ± 0.5℃, and the opposite observed for Vibrio alginolyticus samples and only later post-infection. Comparing the phagocytosis ability results with those obtained from Particle Binding Intensity index important differences concerned mainly confidence levels. The use of phagocytosis ability instead of PBI index provides more accurate results.
Topics: Animals; Fish Diseases; Gram-Negative Bacterial Infections; Hemocytes; Octopodiformes; Phagocytosis; Photobacterium; Temperature; Vibrio alginolyticus
PubMed: 35398307
DOI: 10.1016/j.jip.2022.107754 -
Chembiochem : a European Journal of... Sep 2017A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered...
A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E. coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.
Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigen-Antibody Reactions; Antigens, Tumor-Associated, Carbohydrate; Cancer Vaccines; Chromatography, Thin Layer; Click Chemistry; Enzyme-Linked Immunosorbent Assay; Epitopes; Escherichia coli; Metabolic Engineering; Neisseria; Peptides, Cyclic; Photobacterium; Sialyltransferases; Vaccines, Synthetic
PubMed: 28632300
DOI: 10.1002/cbic.201700240 -
MSphere Feb 2021Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural...
Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-containing peptidase from subsp. (), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA ( lpC-like rotein ) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-d-glutamyl--diaminopimelic acid bond. However, PnpA does not cleave the PG of or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the type II secretion system and degrades the PG of and This suggests that PnpA is used by to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that subsp. , a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.
Topics: Animals; Bacteria; Cell Wall; Endopeptidases; Fishes; Peptidoglycan; Photobacterium
PubMed: 33536321
DOI: 10.1128/mSphere.00736-20 -
Microbes and Environments 2021Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase...
Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.
Topics: Aminopeptidases; Bacteria; Bacterial Proteins; Extracellular Space; Peptides; Phylogeny; Seawater
PubMed: 33716237
DOI: 10.1264/jsme2.ME20150 -
Fish & Shellfish Immunology Oct 2021Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for...
Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5'-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.
Topics: Administration, Oral; Animals; Chitosan; Fish Diseases; Flatfishes; Gram-Negative Bacterial Infections; Nanoparticles; Photobacterium; Vaccines, DNA
PubMed: 34358703
DOI: 10.1016/j.fsi.2021.07.023 -
Journal of Microbiology & Biology... 2018Student-centered teaching allows students to be actively engaged in hands-on, minds-on activities that emphasize creativity and collaboration, enabling them to ask...
Student-centered teaching allows students to be actively engaged in hands-on, minds-on activities that emphasize creativity and collaboration, enabling them to ask questions and design their own investigations to real-world problems. One such problem is water contamination, which causes human health and environmental issues. However, chemical water quality testing for pollutants can be timely and expensive. In addition to chemical testing, researchers have developed assays using unicellular organisms to determine which pollutants are present and in what concentrations. In this three-hour laboratory activity, high school students and undergraduate biology or microbiology students work in pairs to help a fictional company develop a water quality microbioassay. Students design their own laboratory protocols to test the reaction of a bioluminescent bacterial species (i.e., or ) to exposure of common aquatic pollutants such as fertilizer, household cleaners, and motor oil. During this laboratory activity, students apply previously learned components of experimental design, including positive and negative controls, constants, and experimental groups. In addition, students gain experience writing a scientific explanation for a recommendation regarding the bioluminescent bacteria's suitability in a bioassay. Pre- and post-evaluation data revealed that students were successful in achieving the activity's objectives as well as in designing their investigations and writing their protocols using scaffolds within the lesson.
PubMed: 30100954
DOI: 10.1128/jmbe.v19i2.1373 -
International Journal of Molecular... Nov 2022Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities...
Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain's complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.
Topics: Brassica napus; Polyhydroxyalkanoates; Glycerol; Biofuels; Brassica rapa; Genomics; Urea
PubMed: 36430242
DOI: 10.3390/ijms232213754 -
Photochemical & Photobiological... Feb 2020In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we...
In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.
Topics: Amino Acid Sequence; Bacterial Proteins; Cloning, Molecular; Escherichia coli; Evolution, Molecular; Luminescent Measurements; NAD; Oxidoreductases; Photobacterium; Phylogeny; Protein Structure, Secondary; Recombinant Proteins; Sequence Alignment
PubMed: 32002529
DOI: 10.1039/c9pp00435a -
Frontiers in Microbiology 2022Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine...
Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O, 30% CO, red and white meats) and oxygen-free MAP (70% N, 30% CO, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of and was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for . Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for . Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose ( or glycogen ( appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit , heavily reduce growth , and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.
PubMed: 35722325
DOI: 10.3389/fmicb.2022.866629 -
Frontiers in Microbiology 2019The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC)...
The RstAB System Impacts Virulence, Motility, Cell Morphology, Penicillin Tolerance and Production of Type II Secretion System-Dependent Factors in the Fish and Human Pathogen subsp. .
The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen subsp. , a marine bacterium of the family . However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of and mutations in cell fitness and in diverse virulence-related features. Both and mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in and genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. and mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to separate after cell division, whereas at 1% NaCl the mutants exhibited normal phenotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, and mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of subsp. also led to the identification of RstAB-independent potential virulence factors as lipoproteins, sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in subsp. .
PubMed: 31105680
DOI: 10.3389/fmicb.2019.00897