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Disease Models & Mechanisms Aug 2023Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation...
Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute β-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain β-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.
Topics: Animals; Zebrafish; Hyperglycemia; Metronidazole; Diabetes Mellitus; Nitroreductases; Animals, Genetically Modified
PubMed: 37401381
DOI: 10.1242/dmm.050215 -
Applied and Environmental Microbiology Jun 2023Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic spp....
Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic spp. in aquatic ecosystems. To determine the impact of environmental factors on occurrence of pathogenic spp., samples were collected in the Chesapeake Bay, Maryland, during 2009 to 2012 and 2019 to 2022. Genetic markers for Vibrio vulnificus () and Vibrio parahaemolyticus (, , and ) were enumerated by direct plating and DNA colony hybridization. Results confirmed seasonality and environmental parameters as predictors. Water temperature showed a linear correlation with and , and two critical thresholds were observed, an initial increase in detectable numbers (>15°C) and a second increase when maximum counts were recorded (>25°C). Temperature and pathogenic V. parahaemolyticus ( and ) were not strongly correlated; however, the evidence showed that these organisms persist in oyster and sediment at colder temperatures. Salinity (10 to 15 ppt), total chlorophyll (5 to 25 μg/L), dissolved oxygen (5 to 10 mg/L), and pH (8) were associated with increased abundance of and . Importantly, a long-term increase in spp. numbers was observed in water samples between the two collection periods, specifically at Tangier Sound (lower bay), with the evidence suggesting an extended seasonality for these bacteria in the area. Notably, showed a mean positive increase that was ca. 3-fold overall, with the most significant increase observed during the fall. In conclusion, vibriosis continues to be a risk in the Chesapeake Bay region. A predictive intelligence system to assist decision makers, with respect to climate and human health, is warranted. The genus includes pathogenic species that are naturally occurring in marine and estuarine environments globally. Routine monitoring for species and environmental parameters influencing their incidence is critical to provide a warning system for the public when the risk of infection is high. In this study, occurrence of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Chesapeake Bay water, oysters, and sediment samples collected over a 13-year period was analyzed. The results provide a confirmation of environmental predictors for these bacteria, notably temperature, salinity, and total chlorophyll , and their seasonality of occurrence. New findings refine environmental parameter thresholds of culturable species and document a long-term increase in populations in the Chesapeake Bay. This study provides a valuable foundation for development of predicative risk intelligence models for incidence during climate change.
Topics: Animals; Humans; Vibrio parahaemolyticus; Vibrio vulnificus; Chlorophyll A; Ecosystem; Ostreidae; Vibrio Infections; Water
PubMed: 37222620
DOI: 10.1128/aem.00307-23 -
Frontiers in Microbiology 2023Growing concerns exist regarding human ingestion of contaminated seafood that contains biofilms on microplastics (MPs). One of the mechanisms enhancing biofilm related...
Growing concerns exist regarding human ingestion of contaminated seafood that contains biofilms on microplastics (MPs). One of the mechanisms enhancing biofilm related infections in humans is due to biofilm dispersion, a process that triggers release of bacteria from biofilms into the surrounding environment, such as the gastrointestinal tract of human hosts. Dispersal of cells from biofilms can occur in response to environmental conditions such as sudden changes in temperature, pH and nutrient conditions, as the bacteria leave the biofilm to find a more stable environment to colonize. This study evaluated how brief exposures to nutrient starvation, elevated temperature, different pH levels and simulated human media affect and biofilm dispersal and processes on and from low-density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS) MPs. Both species were able to adequately disperse from all types of plastics under most exposure conditions. was able to tolerate and survive the low pH that resembles the gastric environment compared to pH had a significantly ( ≤ 0.05) positive effect on overall biofilm biomass in microplates and cell colonization from PP and PS. pH also had a positive effect on cell colonization from LDPE and PP. However, most biofilm biomass, biofilm cell and dispersal cell densities of both species greatly varied after exposure to elevated temperature, pH, and nutrient starvation. It was also found that certain exposures to simulated human media affected both and biofilm biomass and biofilm cell densities on LDPE, PP and PS compared to exposure to traditional media of similar pH. Cyclic-di-GMP was higher in biofilm cells compared to dispersal cells, but exposure to more stressful conditions significantly increased signal concentrations in both biofilm and dispersal states. Taken together, this study suggests that human pathogenic strains of and can rapidly disperse with high cell densities from different plastic types . However, the biofilm dispersal process is highly variable, species specific and dependent on plastic type, especially under different human body related environmental exposures.
PubMed: 37854331
DOI: 10.3389/fmicb.2023.1236471 -
MicrobiologyOpen Oct 2023Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase β-subunit gene rpoB, has a global impact on bacterial transcription and cell...
Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase β-subunit gene rpoB, has a global impact on bacterial transcription and cell physiology. Previously, we identified a substitution of serine 522 in RpoB (i.e., RpoB ) conferring rifampicin resistance to Vibrio vulnificus, a human food-borne and wound-infecting pathogen associated with a high mortality rate. Transcriptional and physiological analysis of V. vulnificus expressing RpoB showed increased basal transcription of stress-related genes and global virulence regulators. Phenotypically these transcriptional changes manifest as disturbed osmo-stress responses and toxin-associated hypervirulence as shown by reduced hypoosmotic-stress resistance and enhanced cytotoxicity of the RpoB strain. These results suggest that RpoB-linked rifampicin resistance has a significant impact on V. vulnificus survival in the environment and during infection.
Topics: Humans; Rifampin; Vibrio vulnificus; Bacterial Proteins; Mutation; Virulence; DNA-Directed RNA Polymerases
PubMed: 37877661
DOI: 10.1002/mbo3.1379 -
PLoS Biology Aug 2023Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development...
Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.
Topics: Gene Amplification; Anti-Bacterial Agents; Molecular Docking Simulation; Phylogeny; Gram-Negative Bacteria; Gram-Positive Bacteria
PubMed: 37561817
DOI: 10.1371/journal.pbio.3002186 -
Microorganisms Oct 2023() and () are water- and foodborne bacteria that can cause several distinct human diseases, collectively called vibriosis. The success of oyster aquaculture is... (Review)
Review
() and () are water- and foodborne bacteria that can cause several distinct human diseases, collectively called vibriosis. The success of oyster aquaculture is negatively impacted by high abundances. Myriad environmental factors affect the distribution of pathogenic , including temperature, salinity, eutrophication, extreme weather events, and plankton loads, including harmful algal blooms. In this paper, we synthesize the current understanding of ecological drivers of and and provide a summary of various tools used to enumerate and in a variety of environments and environmental samples. We also highlight the limitations and benefits of each of the measurement tools and propose example alternative tools for more specific enumeration of pathogenic and . Improvement of molecular methods can tighten better predictive models that are potentially important for mitigation in more controlled environments such as aquaculture.
PubMed: 37894160
DOI: 10.3390/microorganisms11102502 -
NPJ Vaccines Sep 2023Flagellin, the TLR5 agonist, shows potent adjuvant activities in diverse vaccines and immunotherapies. Vibrio vulnificus flagellin B expressed in eukaryotic cells...
Flagellin, the TLR5 agonist, shows potent adjuvant activities in diverse vaccines and immunotherapies. Vibrio vulnificus flagellin B expressed in eukaryotic cells (eFlaB) could not stimulate TLR5 signaling. Enzymatic deglycosylation restored eFlaB's TLR5 stimulating functionality, suggesting that glycosylation interferes with eFlaB binding to TLR5. Site-directed mutagenesis of N-glycosylation residues restored TLR5 stimulation and adjuvanticity. Collectively, deglycosylated eFlaB may provide a built-in adjuvant platform for eukaryotic-expressed antigens and nucleic acid vaccines.
PubMed: 37752138
DOI: 10.1038/s41541-023-00738-3 -
Biomaterials Jul 2023The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver...
The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4 T, CD8 T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.
Topics: Humans; Animals; Mice; Interleukin-15; Salmonella typhimurium; Neoplasms; Proteins; Immunotherapy; Cell Line, Tumor
PubMed: 37148758
DOI: 10.1016/j.biomaterials.2023.122135 -
MBio Oct 2023Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates...
Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, . Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in .
Topics: Humans; Vibrio vulnificus; Bacterial Proteins; Virulence; Cyclic GMP; Biofilms; Gene Expression Regulation, Bacterial
PubMed: 37800901
DOI: 10.1128/mbio.01536-23