Iron-Utilization System in Vibrio vulnificus M2799

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Visual LAMP Method for Detection of Vibrio vulnificus in Aquatic Products and Environmental Water

Background A visualized, rapid, simple method was developed based on loop-mediated isothermal amplification (LAMP) assay to detect Vibrio vulnificus in aquatic products and aquaculture waters.Results Genomic DNA was extracted from Vibrio vulnificus using the boiling and column extraction methods and optimized primers were used to detect the gyrB gene using a visual LAMP method. The sensitivity of the assay was 10 fg/μL, and the results were stable and reliable. Of 655 aquatic product samples and 558 aquaculture waters samples, the positive rates of Vibrio vulnificus detection were 9.01% and 8.60%, respectively, which is markedly higher than that of the traditional culture identification methods. Conclusion The relatively simple technical requirements, low equipment costs, and rapid detection time make the visual LAMP method for detection of Vibrio vulnificus a convenient choice for field diagnosis in the aquaculture industry.

Evidence for Association of Vibrio Echinoideorum with Tissue Necrosis on Body Shell of the Green Sea Urchin Strongylocentrotus Droebachiensis

“Sea urchin lesion syndrome” is known as sea urchins disease with the progressive development of necrotic epidermal tissue and loss of external organs, including appendages on the outer body surface. Recently, a novel strain, Vibrio echinoideorum has been isolated from the lesions of green sea urchin (Strongylocentrotus droebachiensis), an economically important mariculture species in Norway. V. echinoideorum has not been reported elsewhere in association of with green sea urchin lesion syndrome. Therefore, in this study, an immersion based bacterial challenge experiment was performed to expose sea urchins (wounded and non-wounded) to V. echinoideorum, thereby mimicking a nearly natural host-pathogen interaction under controlled conditions. This infection experiment demonstrated that only the injured sea urchins developed the lesion to a significant degree when exposed to V. echinoideorum. Pure cultures of the employed bacterial strain was recovered from the infected animals and its identity was confirmed by the MALDI-TOF MS spectra profiling. Additionally, the hemolytic phenotype of V. echinoideorum substantiated its virulence potential towards the host, and this was also supported by the cytolytic effect on red spherule cells of sea urchins. Furthermore, the genome sequence of V. echinoideorum was assumed to encode potential virulence genes and were subjected for in silico comparison with the established virulence factors of Vibrio vulnificus and Vibrio tasmaniensis. This comparative virulence profile provided novel insights about virulence genes and their putative functions related to chemotaxis, adherence, invasion, evasion of the host immune system, and damage of host tissue and cells. Thus, it supports the pathogenicity of V. echinoideorum. In conclusion, the interaction of V. echinoideorum with injured sea urchins appears to be essential for the development of lesion syndrome and therefore, revealing its potentiality as an opportunistic pathogen.

Epidemiological and microbiological investigation of the large increase of vibriosis in northern Europe in 2018

Northern European countries and countries bordering the Baltic Sea have witnessed an increase of vibriosis cases during recent heatwaves. Here, we described the epidemiology of vibriosis cases and the genetic diversity of Vibrio isolates from Norway, Sweden, Denmark, Finland, Poland, Estonia, and Latvia in 2018, a year with an exceptionally warm summer. We conducted a retrospective study and analysed demographics, geographic distribution, seasonality, causative species, and severity of non-travel related vibriosis cases in 2018. Data sources included surveillance systems, national laboratory notification databases and/or nationwide surveys to public health microbiology laboratories. Moreover, we performed whole genome sequencing and multilocus sequence typing of available isolates from 2014-2018 to map their genetic diversity. In 2018, we identified 445 non-travel related vibriosis cases in the study countries, which was considerably higher than the median of 126 cases between 2014-2017 (range: 87-272). The main reported mode of transmission was exposure to seawater. We observed a species-specific geographic disparity of vibriosis cases across the Nordic-Baltic region. Developing severe vibriosis was associated with infections caused by Vibrio vulnificus (adjOR: 17.2; 95% CI: 3.3-90.5) or Vibrio parahaemolyticus (adjOR: 2.1; 95% CI: 1.0-4.5), being ≥65 years of age (65-79 years, adjOR: 3.9; 95% CI: 1.7-8.7; 80+ years, adjOR: 15.5; 95% CI: 4.4-54.3) or acquiring infections during summer (adjOR: 5.1; 95% CI: 2.4-10.9). Although phylogenetic analysis revealed diversity between Vibrio isolates, two V. vulnificus clusters (<10 SNPs) were identified. Study countries could benefit from establishing a shared sentinel surveillance system for vibriosis to address this emerging public health issue.

Pre-injection of Zebrafish (Danio rerio) tnfb Polyclonal Antibody Decreases the Mortality of Vibrio vulnificus Infected Zebrafish

Tumor necrosis factor (TNF) plays an important role in an inflammatory cytokine storm. Over-secretion of TNF by the host in response to infection aggravates the disease. TNF expression level is positively correlated with the mortality caused by some bacterial infections. Therefore, using TNF antibody may alleviate the inflammation to resist bacterial infections. The function of fish TNF-b antibody in bacterial infection is still unclear. In this study, infection models of Vibrio vulnificus FJ03-X2 strain with high pathogenicity and strong virulence were established in zebrafish (Danio rerio) fibroblast cell line (ZF4 cells) and zebrafish. Zebrafish tnfb (Zetnf-b) gene was cloned and expressed by Escherichia coli BL21 (DE3), and Zetnf-b polyclonal antibody was prepared. Pre-injection of Zetnf-b polyclonal antibody and AG-126 before infecting with V. vulnificus could increase the survival rate of zebrafish by 36.6 and 46.7%, respectively. Pre-injection of Zetnf-b polyclonal antibody could effectively decrease the mortality of zebrafish infected by V. vulnificus. Thus, TNF polyclonal antibody therapy could be considered as an effective strategy to control V. vulnificus in fish.

Flagellin synergistically enhances anti-tumor effect of EGFRvIII peptide in a glioblastoma-bearing mouse brain tumor model

Purpose Glioblastoma (GBM) is the most aggressive type of brain tumor with heterogeneity and strong invasive ability. Treatment of GBM has not improved significantly despite the progress of immunotherapy and classical therapy. Epidermal growth factor receptor variant III (EGFRvIII), one of GBM-associated mutants, is regarded as an ideal therapeutic target in EGFRvIII-expressed GBM patients because it is a tumor-specific receptor expressed only in tumors. Flagellin B (FlaB) originated from Vibrio vulnificus is known as a strong adjuvant that enhance innate and adaptive immunity in various vaccine models. This study performed whether FlaB synergistically could enhance the anti-tumor effect of EGFRvIII peptide (PEGFRvIII). Methods EGFRvIII-GL261/Fluc cells were used for glioblastoma-bearing mouse brain model and cell-bearing mice were inoculated with PBS, FlaB alone, PEGFRvIII alone, and PEGFRvIII plus FlaB. Survival rate through tumor growth was investigated by MRI. T cell population was examined by flow cytometry analysis. Both cleaved caspase-3 and CD8+ lymphocytes were shown by immunohistochemistry (IHC) staining. Results The PEGFRvIII plus FlaB group showed delayed tumor growth and increased survival rate when compared to other treatment groups. As an evidence of apoptosis, cleaved caspase-3 expression and DNA disruption were more increased in the PEGFRvIII plus FlaB group than in other groups. In addition, the PEGFRvIII plus FlaB group showed more increased CD8+ T cells and decreased Treg cells than other treatment groups in the brain. Conclusion FlaB can enhance the anti-tumor effect of PEGFRvIII by increasing CD8+ T cell response in a mouse brain GBM model.

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Bacterial human pathogens secrete initially inactive nucleotidyl cyclases that become potent enzymes by binding to actin inside eukaryotic host cells. The underlying molecular mechanism of this activation is, however, unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus bound to their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo-state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. Other nucleotidyl cyclase toxins that bind to calmodulin rather than actin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is conserved in these enzymes, albeit the different activator.