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Frontiers in Microbiology 2023Lead (Pb) is a hazardous pollutant in water environments that can cause significant damage to aquatic animals and humans. In this study, crucian carp () were exposed to...
Lead (Pb) is a hazardous pollutant in water environments that can cause significant damage to aquatic animals and humans. In this study, crucian carp () were exposed to waterborne Pb for 96 h; then, histopathological analysis, quantitative qPCR analysis, and 16S high-throughput sequencing were performed to explore the effects of Pb on intestinal bioaccumulation, structural damage, oxidative stress, immune response, and microbiota imbalance of . After Pb exposure, the intestinal morphology was obviously damaged, including significantly increasing the thickness of the intestinal wall and the number of goblet cells and reducing the depth of intestinal crypts. Pb exposure reduced the mRNA expressions of and while significantly elevated the level of , and α. Furthermore, 16S rRNA analysis showed that the Shannon and Simpson indices decreased at 48 h after Pb exposure, and the abundance of pathogenic bacteria (Erysipelotrichaceae, Weeksellaceae, and Vibrionaceae) increased after Pb exposure. In addition, the correlation network analysis found that were negatively correlated with and positively correlated with . Functional prediction analysis of bacteria speculated that the change in intestinal microbiota led to the PPAR signaling pathway and peroxisome function of the intestine of crucian carp was increased, while the immune system and membrane transport function were decreased. Finally, canonical correlation analysis (CCA) found that there were correlations between the intestinal microbiota, morphology, antioxidant factors, and immune factors of crucian carp after Pb exposure. Taken together, our results demonstrated that intestinal flora dysbiosis, morphological disruption, oxidative stress, and immune injury are involved in the toxic damage of Pb exposure to the intestinal structure and function of crucian carp. Meanwhile, Pb exposure rapidly increased the abundance of pathogenic bacteria, leading to intestinal disorders, further aggravating the damage of Pb to intestinal structure and function. These findings provide us a basis for the link between gut microbiome changes and heavy metal toxicity, and gut microbiota can be used as biomarkers for the evaluation of heavy metal pollution in future.
PubMed: 37731918
DOI: 10.3389/fmicb.2023.1239323 -
Microbiology Spectrum Apr 2024Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and...
Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O)-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and characterization of genetic circuits encoding the O-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of , a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O-independant flavin-binding fluorescent protein. We demonstrated that cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that NS1, a facultative anaerobe, is fluorescent in the absence of O when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O-independent fluorescence will open new perspective to study anaerobic processes.
Topics: Green Fluorescent Proteins; Bacteria; Flavins; Oxygen; Vibrio
PubMed: 38441526
DOI: 10.1128/spectrum.04091-23 -
Nucleic Acids Research Apr 2024CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of...
CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.
Topics: Adenine Nucleotides; CRISPR-Associated Proteins; CRISPR-Cas Systems; Membrane Proteins; Nucleotides, Cyclic; Second Messenger Systems; Bacterial Proteins; Vibrio cholerae
PubMed: 38471818
DOI: 10.1093/nar/gkae167 -
Virulence Dec 2023is a waterborne bacterium that primarily infects the human intestine and causes cholera fatality. Quorum sensing (QS) negatively regulates the expression of virulence...
is a waterborne bacterium that primarily infects the human intestine and causes cholera fatality. Quorum sensing (QS) negatively regulates the expression of virulence gene. However, the primary associated mechanisms remain undetermined. This investigation identified a new QS regulator from the TetR family, LuxT, which increases virulence by directly inhibiting expression. HapR is a master QS regulator that suppresses virulence cascade expression. The expression of increased 4.8-fold in the small intestine of infant mice than in Luria-Bertani broth. Δ mutant strain revealed a substantial defect in the colonizing ability of the small intestines. At low cell densities, the expression level of was upregulated by deletion, suggesting that LuxT can suppress transcription. The electrophoretic mobility shift analysis revealed that LuxT directly binds to the promoter region. Furthermore, expression was upregulated by the two-component system ArcB/ArcA, which responses to changes in oxygen levels in response to the host's small intestine's anaerobic signals. In conclusion, this research reveals a novel cell density-mediated virulence regulation pathway and contributes to understanding the complex association between virulence and QS signals. This evidence furnishes new insights for future studies on pathogenic mechanisms.
Topics: Animals; Humans; Mice; Vibrio cholerae; Quorum Sensing; Virulence; Cholera; Gene Expression Regulation, Bacterial; Bacterial Proteins
PubMed: 37908129
DOI: 10.1080/21505594.2023.2274640 -
Scientific Reports Mar 2024It is widely believed that a significant portion of the gut microbiota, which play crucial roles in overall health and disease, originates from the food we consume....
It is widely believed that a significant portion of the gut microbiota, which play crucial roles in overall health and disease, originates from the food we consume. Sashimi is a type of popular raw seafood cuisine. Its microbiome, however, remained to be thoroughly explored. The objective of this study is to explore the microbiome composition in sashimi at the time when it is served and ready to be eaten. Specifically, our tasks include investigating the diversity and characteristics of microbial profiles in sashimi with respect to the fish types. We utilized the Sanger-sequencing based DNA barcoding technology for fish species authentication and next-generation sequencing for sashimi microbiome profiling. We investigated the microbiome profiles of amberjack, cobia, salmon, tuna and tilapia sashimi, which were all identified using the MT-CO1 DNA sequences regardless of their menu offering names. Chao1 and Shannon indexes, as well as Bray-Curtis dissimilarity index were used to evaluate the alpha and beta diversities of sashimi microbiome. We successfully validated our previous observation that tilapia sashimi has a significantly higher proportions of Pseudomonas compared to other fish sashimi, using independent samples (P = 0.0010). Salmon sashimi exhibited a notably higher Chao1 index in its microbiome in contrast to other fish species (P = 0.0031), indicating a richer and more diverse microbial ecosystem. Non-Metric Multidimensional Scaling (NMDS) based on Bray-Curtis dissimilarity index revealed distinct clusters of microbiome profiles with respect to fish types. Microbiome similarity was notably observed between amberjack and tuna, as well as cobia and salmon. The relationship of microbiome similarity can be depicted as a tree which resembles partly the phylogenetic tree of host species, emphasizing the close relationship between host evolution and microbial composition. Moreover, salmon exhibited a pronounced relative abundance of the Photobacterium genus, significantly surpassing tuna (P = 0.0079), observed consistently across various restaurant sources. In conclusion, microbiome composition of Pseudomonas is significantly higher in tilapia sashimi than in other fish sashimi. Salmon sashimi has the highest diversity of microbiome among all fish sashimi that we analyzed. The level of Photobacterium is significantly higher in salmon than in tuna across all the restaurants we surveyed. These findings provide critical insights into the intricate relationship between the host evolution and the microbial composition. These discoveries deepen our understanding of sashimi microbiota, facilitating our decision in selecting raw seafood.
Topics: Animals; Phylogeny; Microbiota; Gastrointestinal Microbiome; Salmon; Tuna; Seafood; Photobacterium; Pseudomonas
PubMed: 38443405
DOI: 10.1038/s41598-024-55938-1 -
Proceedings of the National Academy of... Sep 2023the causative agent of the disease cholera, is responsible for multiple pandemics. binds to and colonizes the gastrointestinal tract within the human host, as well as...
the causative agent of the disease cholera, is responsible for multiple pandemics. binds to and colonizes the gastrointestinal tract within the human host, as well as various surfaces in the marine environment (e.g., zooplankton) during interepidemic periods. A large adhesin, the Flagellar Regulated Hemagglutinin A (FrhA), enhances binding to erythrocytes and epithelial cells and enhances intestinal colonization. We identified a peptide-binding domain (PBD) within FrhA that mediates hemagglutination, binding to epithelial cells, intestinal colonization, and facilitates biofilm formation. Intriguingly, this domain is also found in the ice-binding protein of the Antarctic bacterium , where it mediates binding to diatoms. Peptide inhibitors of the PBD inhibit binding to human cells as well as to diatoms and inhibit biofilm formation. Moreover, the PBD inserted into FrhA allows to bind human cells and colonize the intestine and also enhances biofilm formation, demonstrating the interchangeability of the PBD from these bacteria. Importantly, peptide inhibitors of PBD reduce intestinal colonization in infant mice. These studies demonstrate how uses a PBD shared with a diatom-binding Antarctic bacterium to facilitate intestinal colonization in humans and biofilm formation in the environment.
Topics: Animals; Humans; Infant; Mice; Bacteria; Cell Aggregation; Diatoms; Gastrointestinal Tract; Intestines; Vibrio cholerae
PubMed: 37729203
DOI: 10.1073/pnas.2308238120 -
Marine Pollution Bulletin Aug 202316S rRNA gene sequencing and bacteria- and genus-specific quantitative PCR was used to profile microbial communities and their associated functions in water, live feed...
16S rRNA gene sequencing and bacteria- and genus-specific quantitative PCR was used to profile microbial communities and their associated functions in water, live feed (microalgae, Artemia, and rotifer), and European sea bass and gilthead sea bream larvae from hatcheries in Greece and Italy. The transfer to larvae of genus containing potential pathogens of fish was more likely with Artemia and rotifer than with microalgae or water, irrespective of geographic location. The presence of potentially pathogenic bacteria (Vibrio and Pseudoalteromonas) in the core microbiota of water, live feed, and fish larvae, the enrichment of different bacterial resistance pathways and biofilm formation, and the overall low beneficial bacteria load during larval ontogeny emphasizes the risk for disease outbreaks. The present data characterizing microbiota in commercial aquaculture hatcheries provides a baseline for the design of strategies to manage disease and to model or remediate potential adverse environmental impacts.
Topics: Animals; RNA, Ribosomal, 16S; Aquaculture; Microbiota; Rotifera; Vibrio; Larva; Water
PubMed: 37441915
DOI: 10.1016/j.marpolbul.2023.115218 -
ELife Jul 2023Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived 'autoinducer' signalling molecules, that accumulate in...
Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived 'autoinducer' signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In , quorum-sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in . Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows to regulate subsets of genes during the transition between aquatic environments and the human host.
Topics: Humans; Vibrio cholerae; Quorum Sensing; Repressor Proteins; Bacterial Proteins; Transcription Factors; Cyclic AMP Receptor Protein; Gene Expression Regulation, Bacterial
PubMed: 37410076
DOI: 10.7554/eLife.86699 -
Cell Reports Feb 2024To maintain an effective barrier, intestinal progenitor cells must divide at a rate that matches the loss of dead and dying cells. Otherwise, epithelial breaches expose...
To maintain an effective barrier, intestinal progenitor cells must divide at a rate that matches the loss of dead and dying cells. Otherwise, epithelial breaches expose the host to systemic infection by gut-resident microbes. Unlike most pathogens, Vibrio cholerae blocks tissue repair by arresting progenitor proliferation in the Drosophila model. At present, we do not understand how V. cholerae circumvents such a critical antibacterial defense. We find that V. cholerae blocks epithelial repair by activating the growth inhibitor bone morphogenetic protein (BMP) pathway in progenitors. Specifically, we show that interactions between V. cholerae and gut commensals initiate BMP signaling via host innate immune defenses. Notably, we find that V. cholerae also activates BMP and arrests proliferation in zebrafish intestines, indicating an evolutionarily conserved link between infection and failure in tissue repair. Our study highlights how enteric pathogens engage host immune and growth regulatory pathways to disrupt intestinal epithelial repair.
Topics: Animals; Zebrafish; Anti-Bacterial Agents; Bone Morphogenetic Proteins; Drosophila; Vibrio cholerae; Cell Proliferation
PubMed: 38340318
DOI: 10.1016/j.celrep.2024.113750 -
Microbiome Jul 2023For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one...
BACKGROUND
For more than a century, the Koch's postulates have been the golden rule for determining the causative agents in diseases. However, in cases of multiple pathogens-one disease, in which different pathogens can cause the same disease, the selection of microorganisms that regress infection is hard when Koch's postulates are applied. Microbiome approaches can obtain relatively complete information about disease-related microorganisms and can guide the selection of target microorganisms for regression infection. In the present study, whitish muscle syndrome (WMS) of Scylla paramamosain, which has typical symptoms with whitish muscle and blackened hemolymph was used as an example to establish a new research strategy that integrates microbiome approaches and Koch's postulates to determinate causative agents of multiple pathogens-one disease.
RESULTS
Microbiome results revealed that Aeromonas, Acinetobacter, Shewanella, Chryseomicrobium, Exiguobacterium, Vibrio and Flavobacterium, and Kurtzmaniella in hemolymph were bacterial and fungal indicators for WMS. A total of 23 bacteria and 14 fungi were isolated from hemolymph and muscle tissues, and among the bacteria, Shewanella chilikensis, S. xiamenensis, Vibrio alginolyticus, S. putrefaciens, V. fluvialis, and V. parahaemolyticus were present in hemolymph and/or muscle tissues in each WMS crab, and the last three species were also present in three Healthy crabs. The target bacteria and fungi were further screened to regression infections based on two criteria: whether they belonged to the indicator genera for WMS, whether they were isolated from both hemolymph and muscle tissues in most WMS crabs. Only S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria. The six bacteria that met both two criteria and six fungi and another bacterium that unmatched any of two criteria were used to perform regression infection experiments based on Koch's postulates. S. chilikensis, S. putrefaciens, S. xiamenensis, V. alginolyticus, V. fluvialis, and V. parahaemolyticus met both two criteria, and the results indicate that they cause WMS in crabs independently.
CONCLUSIONS
This study fully demonstrated that our research strategy that integrates the microbiome and Koch's postulates can maximize the ability to catch pathogens in one net for the situation of multiple pathogens-one disease. Video Abstract.
Topics: Animals; Brachyura; Vibrio; Microbiota; Muscles
PubMed: 37475003
DOI: 10.1186/s40168-023-01570-6