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Fish & Shellfish Immunology Jul 2024Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential...
Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two β-domains (β2β1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against HO-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.
Topics: Animals; Metallothionein; Gastropoda; Oxidative Stress; Vibrio parahaemolyticus; Immunity, Innate; Novirhabdovirus; Gene Expression Regulation; Amino Acid Sequence; Phylogeny; Sequence Alignment; Listeria monocytogenes; Mice; Gene Expression Profiling; RAW 264.7 Cells; Metals, Heavy; Water Pollutants, Chemical
PubMed: 38777254
DOI: 10.1016/j.fsi.2024.109645 -
Applied and Environmental Microbiology Jun 2024CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific...
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible and genes into the genome using Tn transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple species: , , , , and . We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in species.
Topics: Vibrio; Vibrio vulnificus; Vibrio parahaemolyticus; Gene Expression Regulation, Bacterial; CRISPR-Cas Systems; Vibrio cholerae; Clustered Regularly Interspaced Short Palindromic Repeats; Bacterial Proteins; Gene Knockdown Techniques; Aliivibrio fischeri
PubMed: 38775491
DOI: 10.1128/aem.00065-24 -
Scientific Reports May 2024Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp....
Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp. Functional studies showed that SB dose-dependently increased shrimp body weight and decreased feed conversion ratio. We found that SB protected against Vibrio parahaemolyticus as evidenced by survival rate, bacterial load, and hepatopancreatic pathology in shrimp. Finally, we explored the likely mechanism by which SB affects growth performance and vibriosis in shrimp. The 16S rRNA sequencing data showed that SB increased 6 probiotic genera and decreased 6 genera of pathogenic bacteria in shrimp. Among these, SB increased the proportion of Lactobacillus johnsonii and decreased that of V. parahaemolyticus in shrimp guts. To dissect the relationship among SB, Lactobacillus and Vibrio, we investigated the in vitro regulation of Lactobacillus and Vibrio by SB. SB at ≥ 0.25 μg/mL promoted L. johnsonii growth. Additionally, L. johnsonii and its supernatant could inhibit V. parahaemolyticus. Furthermore, SB could up-regulate five anti-Vibrio metabolites of L. johnsonii, which caused bacterial membrane destruction. In parallel, we identified 3 fatty acids as active compounds from SB. Overall, this work demonstrated that SB improved growth performance and vibriosis protection in shrimp via the regulation of gut microbiota.
Topics: Animals; Penaeidae; Vibrio parahaemolyticus; Vibrio Infections; Lactobacillus; RNA, Ribosomal, 16S; Vibrio; Probiotics
PubMed: 38773245
DOI: 10.1038/s41598-024-62436-x -
The Science of the Total Environment Jul 2024The widespread presence of microplastics (MP) in water represents an environmental problem, not only because of the harmful effects of their size and potential to vector...
The widespread presence of microplastics (MP) in water represents an environmental problem, not only because of the harmful effects of their size and potential to vector other pollutants, but also because of the release of additives, degradation products and residues contained in the polymer matrix. The latter includes metallic catalysts, which are often overlooked. This study focuses on the photo-aging of polypropylene (PP) and the resulting structural changes that promote its fragmentation microplastics (PP-MPs) and release of metals, as well as the resulting toxicity of leachates and their potential to inhibit biodegradation of organics in water. The pristine, photo-aged and waste PP are ground under the same regime to assess susceptibility to fragmentation. Obtained PP-MPs are submitted to leaching tests; the release of organics and metals is monitored by Total Organic Carbon (TOC) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis, respectively. The leachates are assessed for their toxicity against Vibrio fischeri, Daphnia magna and Pseudokirchneriella subcapitata and their influence on the biodegradability of the glucose solution. Photo-aging induced changes in the crystallinity and morphology of the PP and manifested in the abundance of smaller MPs, as revealed by the particle size distribution. In the case of pristine PP, all particles were > 100 μm in size, while aged PP yielded significant mass fraction of MPs <100 μm. The toxicity of leachates from aged PP-MPs is higher than that of pristine and exhibits a positive correlation with portion of metals released. The biodegradability of glucose is strongly inhibited by PP-MPs leachates containing a mixture of metals in trace concentrations.
Topics: Water Pollutants, Chemical; Microplastics; Biodegradation, Environmental; Polypropylenes; Daphnia; Metals; Aliivibrio fischeri; Animals
PubMed: 38772480
DOI: 10.1016/j.scitotenv.2024.173344 -
Aquatic Toxicology (Amsterdam,... Jul 2024As one of the main components of marine pollution, microplastics (MPs) inevitably enter the mussel aquaculture environment. At the same time, pathogenic bacteria,...
As one of the main components of marine pollution, microplastics (MPs) inevitably enter the mussel aquaculture environment. At the same time, pathogenic bacteria, especially pathogens such as Vibrio, can cause illness outbreaks, leading to large-scale death of mussels. The potential harm of MPs and pathogenic bacteria to bivalve remains unclear. This study designed two experiments (1) mussels (Mytilus galloprovincialis) were exposed to 100 particles/L or 1,000 particles/L polymethyl methacrylate (PMMA, 17.01 ± 6.74 μm) MPs and 1 × 10 CFU/mL Vibrio parahaemolyticus at the same time (14 days), and (2) mussels were exposed to 100 particles/L or 1,000 particles/L MPs for a long time (30 days) and then exposed to 1 × 10 CFU/mL V. parahaemolyticus to explore the effects of these two stresses on the mussel immune system. The results showed that after the combined exposure of V. parahaemolyticus and MPs, the lysosomal membrane stability of hemocytes decreased, lysozyme activity was inhibited, and hemocytes were induced to produce more lectins and defensins to fight pathogenic invasion. Long-term exposure to MPs caused a large amount of energy consumption in mussels, inhibited most of the functions of humoral immunity, increased the risk of mussel infection with pathogenic bacteria, and negatively affected mussel condition factor, the number of hemocytes, and the number of byssuses. Mussels may allocate more energy to deal with MPs and pathogenic bacterial infections rather than for growth. Above all, MPs exposure can affect mussel immune function or reduce its stress resistance, which in turn has an impact on mollusk farming.
Topics: Animals; Mytilus; Microplastics; Vibrio parahaemolyticus; Water Pollutants, Chemical; Hemocytes; Muramidase; Immune System
PubMed: 38768528
DOI: 10.1016/j.aquatox.2024.106959 -
BioRxiv : the Preprint Server For... May 2024Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known...
UNLABELLED
Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen were unknown. In this study, we showed that to alleviate osmotic stress biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not -inositol. -inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified -inositol ( ) catabolism clusters in and other and species. Growth pattern analysis demonstrated that utilized -inositol as a sole carbon source, with a short lag time of 3 h. An deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on -inositol. Within the clusters were an MFS-type ( and an ABC-type ( transporter and analyses showed that both transported -inositol. IolG and IolA phylogeny among species showed different evolutionary histories indicating multiple acquisition events. Outside of , IolG was most closely related to IolG from a small group of fish and human pathogens and species. However, IolG from hypervirulent strains clustered with IolG from and divergently from and plant pathogens. The cluster was also present within and of which many species were associated with marine flora and fauna.
IMPORTANCE
Host associated bacteria such as encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that -inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of -inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of -inositol metabolism is complex, acquired multiple times in but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.
PubMed: 38766061
DOI: 10.1101/2024.01.16.575920 -
International Journal of Food... Jun 2024This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived...
This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen.
Topics: China; Drug Resistance, Multiple, Bacterial; Food Microbiology; Anti-Bacterial Agents; Animals; Humans; Microbial Sensitivity Tests; Cholera; Vibrio cholerae; Vibrio cholerae non-O1; Plasmids
PubMed: 38759293
DOI: 10.1016/j.ijfoodmicro.2024.110734 -
Fish & Shellfish Immunology Jul 2024C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp...
C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca in invertebrates.
Topics: Animals; Penaeidae; Lectins, C-Type; Amino Acid Sequence; Immunity, Innate; Vibrio; Phylogeny; Arthropod Proteins; Sequence Alignment; White spot syndrome virus 1; Base Sequence; Calcium; Gene Expression Regulation; Gene Expression Profiling
PubMed: 38754650
DOI: 10.1016/j.fsi.2024.109638 -
Analytical Chemistry May 2024Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a...
Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen , the marine symbiont , and the opportunistic pathogen PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.
Topics: Cyclic GMP; Biofilms; Pseudomonas aeruginosa; Vibrio cholerae; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Aliivibrio fischeri
PubMed: 38752543
DOI: 10.1021/acs.analchem.3c04687 -
Scientific Reports May 2024Biofloc (BF) stands out as a promising system for sustainable shrimp farming. Optimizing various culture conditions, such as stocking density, carbohydrate source, and...
Influence of stocking density on the growth, immune and physiological responses, and cultivation environment of white-leg shrimp (Litopenaeus vannamei) in biofloc systems.
Biofloc (BF) stands out as a promising system for sustainable shrimp farming. Optimizing various culture conditions, such as stocking density, carbohydrate source, and feeding management, is crucial for the widespread adoption of the BF system. This study compares the growth performance of white-leg shrimp (Litopenaeus vannamei) in culture ponds at low density (LD) with 50 organisms/m and high density (HD) with 200 organisms/m. Post-larvae of white-leg shrimp were stocked for 16 weeks in both LD and HD groups. The LD group exhibited a superior survival rate, growth rate, and feed consumption compared to the HD group. The BF from the LD system recorded a significantly higher protein content (16.63 ± 0.21%) than the HD group (15.21 ± 0.34%). Heterotrophic bacterial counts in water did not significantly differ with stocking density. However, Vibrio count in water samples was higher in the HD group (3.59 ± 0.35 log CFU/mL) compared to the LD group (2.45 ± 0.43 log CFU/mL). The whole shrimp body analysis revealed significantly higher protein and lipid content in the LD group. In contrast, the total aerobic bacterial count in shrimp from the HD group was high, with the identification of Salmonella enterica ssp. arizonae. Additionally, Vibrio counts in shrimp samples were significantly higher in the HD group (4.63 ± 0.32 log CFU/g) compared to the LD group (3.57 ± 0.22 log CFU/g). The expression levels of immune-associated genes, including prophenoloxidase, transglutaminase, penaiedin 3, superoxide dismutase, lysozyme, serine proteinase, and the growth-related gene ras-related protein (rap-2a), were significantly enhanced in the LD group. Conversely, stress-related gene expression increased significantly in the HD group. Hepatopancreases amylase, lipase, and protease were higher in the LD group, while trypsin activity did not differ significantly. Antioxidant enzyme activity (catalase, glutathione, glutathione peroxidase, and superoxide dismutase) significantly increased in the LD group. The histological structure of hepatopancreas, musculature, and female gonads remained similar in both densities. However, negative effects were observed in the gills' histology of the HD group. These results suggest that increasing stocking density is associated with significantly negative biological, microbial, and physiological effects on white-leg shrimp under the BF system.
Topics: Animals; Penaeidae; Aquaculture; Vibrio; White
PubMed: 38750082
DOI: 10.1038/s41598-024-61328-4