Journal: Scientific Reports

Article Title: Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

doi: 10.1038/s41598-019-39554-y

Figure Lengend Snippet: High-throughput screening for HlyU inhibitors. ( a ) Schematic demonstration of high-throughput screening of small molecules. An E. coli reporter strain contains pKK1306 expressing HlyU under arabinose-inducible promoter P BAD and pZW1608 carrying the luxCDABE genes under HlyU-repressed promoter P VVMO6_00539 . ( b – d ) Each bar represents RLU of the E. coli reporter strain ( b ) and V. vulnificus reporter strains containing pZW1608 ( c ) or pZW1609 ( d ) in the presence of hit molecules as indicated. Error bars represent the standard deviation (SD) from biological triplicates. Statistical significance was determined by multiple comparisons after one-way analysis of variance (ANOVA) (*** p < 0.0005). 1025E12, 1030B04, and 1040E12, hit molecules; Positive, RLUs from E. coli without arabinose ( b ) or V. vulnificus hlyU mutant ( c , d ); Negative, RLUs from E. coli with arabinose ( b ) or V. vulnificus wild type ( c , d ); RLU, relative luminescence unit.

Article Snippet: HlyU and DnaK in the cell lysates were detected using rabbit anti- V. vulnificus HlyU antibody and mouse anti- E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis.

Techniques: High Throughput Screening Assay, Expressing, Standard Deviation, Mutagenesis

Journal: Scientific Reports

Article Title: Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

doi: 10.1038/s41598-019-39554-y

Figure Lengend Snippet: CM14 inhibits the HlyU activity without affecting V. vulnificus growth. ( a ) The chemical structure of CM14, N -(4-oxo-4H-thieno[3, 4-c]chromen-3-yl)-3-phenylprop-2-ynamide. ( b ) The EC 50 of CM14 inhibiting HlyU activity was calculated as described in the Methods section from three independent experiments. ( c ) Clear lysate samples of the wild type and hlyU mutant cells grown along with various concentrations of CM14 or DMSO (control) were resolved by SDS-PAGE, after which HlyU and DnaK were detected by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. Representative images of Western blot for HlyU and DnaK are shown, and the full-length blots are presented in Supplementary Figure . ( d ) Growth of the V. vulnificus strains along with various concentrations of CM14 or 2% DMSO (control) was monitored at 1 h intervals using a microplate reader. ( e ) Cytotoxicity was determined using LDH activities released from INT-407 cells incubated at 37 °C for 3 h with various concentrations of CM14, the wild-type V. vulnificus (at an MOI of 10), or 2% DMSO (control). The cytotoxicity was expressed using the LDH activity from the cells completely lysed by 5% Triton X-100 as 100%. Error bars represent the SD from the representative of three independent experiments. Statistical significance was determined by one-way ANOVA ( e ) (*** p < 0.0005; ns , not significant; ND, not detected). WT, wild type; hlyU , hlyU mutant; MOI, multiplicity of infection.

Article Snippet: HlyU and DnaK in the cell lysates were detected using rabbit anti- V. vulnificus HlyU antibody and mouse anti- E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis.

Techniques: Activity Assay, Mutagenesis, SDS Page, Western Blot, Incubation, Infection

Journal: Scientific Reports

Article Title: Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

doi: 10.1038/s41598-019-39554-y

Figure Lengend Snippet: Effects of CM14 on the virulence-related phenotypes of V. vulnificus . ( a , b ) The V. vulnificus strains grown along with CM14 as indicated or DMSO (control) were harvested and fractionated for further analyses. ( a ) The transcript levels of vvhA , rtxA , and plpA in the total RNA of the cells were quantified by qRT-PCR and expressed using each transcript level of the wild type in the presence of DMSO as 1. ( b ) Hemolytic activities of the culture supernatants were determined against human erythrocytes and expressed using complete hemolysis by 5% Triton X-100 as 100%. ( c ) Morphological changes of HeLa cells infected with the V. vulnificus strains along with CM14 (50 μM) or DMSO (control) were photographed. Scale bars, 100 μm. ( d ) Cytotoxicity was determined using LDH activities released from INT-407 cells infected with the V. vulnificus strains along with CM14 as indicated and expressed using the LDH activity from the cells completely lysed by 5% Triton X-100 as 100%. Error bars represent the SD from three independent experiments ( a , b ) and from the representative of three independent experiments ( d ). Statistical significance was determined by the Student’s t -test ( a ) and by one-way ANOVA ( b , d ) (*** p < 0.0005; ** p < 0.005; * p < 0.05; ns , not significant). WT, wild type; hlyU , hlyU mutant.

Article Snippet: HlyU and DnaK in the cell lysates were detected using rabbit anti- V. vulnificus HlyU antibody and mouse anti- E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis.

Techniques: Quantitative RT-PCR, Infection, Activity Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

doi: 10.1038/s41598-019-39554-y

Figure Lengend Snippet: Effects of CM14 on the survival, pathophysiological changes, and inflammatory responses of mice infected with V. vulnificus . ( a ) Survival of the mice anesthetized with isoflurane and subcutaneously injected with wild type ( n = 10), wild type with CM14 ( n = 10), hlyU mutant ( n = 5) at doses of 7.5 × 10 5 colony forming unit (CFU), or CM14 alone ( n = 5, control). ( b – e ) The mice, injected as described in ( a ), were sacrificed at 7 h post infection to obtain blood and skin tissue samples. ( b ) The levels of TP, ALB, AST, ALT, BUN, and CREA in the blood plasma of each group [WT + DMSO ( n = 10), WT + CM14 ( n = 8), hlyU + DMSO ( n = 6), DMSO ( n = 6, control), and CM14 ( n = 6, control)] were determined by blood biochemical analysis. The data represent the mean ± SD. Statistical significance was determined by multiple comparisons after one-way ANOVA (* p < 0.05 relative to PBS + DMSO; # p < 0.05 relative to WT + DMSO). ( c , d ) The cytokine levels of IL-1β ( c ) and IL-6 ( d ) in the blood plasma of each group ( n = 7) were quantified by enzyme-linked immunosorbent assay (ELISA). ( e ) Infiltration of macrophages at the injection sites was determined using skin tissue samples that were immune-stained with F4/80 antibody (for macrophages, red) and DAPI (for nucleus, blue) for counter staining. The percentage of F4/80 + cells in DAPI + cells was analyzed by using MetaMorph software. Scale bars, 10 μm ( n = 4). Error bars represent the SD. Statistical significance was determined by log rank test ( a ) and by multiple comparisons after one-way ANOVA ( c – e ) (**** p < 0.0001; *** p < 0.0005; ** p < 0.005; * p < 0.05; ns , not significant; ND, not detected). WT, wild type; hlyU , hlyU mutant.

Article Snippet: HlyU and DnaK in the cell lysates were detected using rabbit anti- V. vulnificus HlyU antibody and mouse anti- E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis.

Techniques: Infection, Injection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining, Software

Journal: Scientific Reports

Article Title: Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species

doi: 10.1038/s41598-019-39554-y

Figure Lengend Snippet: Proposed molecular mechanism underlying the CM14-mediated inhibition of HlyU binding to target DNA. A possible reaction mechanism for the Cys30 modification of HlyU by CM14 is shown in a blue dashed box on the right. First, the sulfur atom of Cys96 of HlyU reacts with a carbon atom (asterisk) of CM14, a Michael reaction acceptor site. Then, a sulfur atom of Cys30 of HlyU attacks a carbonyl carbon of CM14 and releases an amine group with bulky rings. Subsequently, a nucleophile (Nu, e.g. His92) around the reaction site cleaves the sulfur-carbon bond between Cys96 and the remaining part of CM14, protonating the carbon to create the carbon-carbon double bond. The remaining part of CM14 (represented by a green rounded box) is covalently linked to the sulfur atom of Cys30. Active HlyU can bind to target promoter DNA, leading to the production of virulence factors and making V. vulnificus fully virulent. In contrast, inactivation of HlyU by CM14 inhibits the DNA binding of HlyU, resulting in reduced expression of HlyU-regulated virulence genes. These events eventually attenuate the virulence of V. vulnificus .

Article Snippet: HlyU and DnaK in the cell lysates were detected using rabbit anti- V. vulnificus HlyU antibody and mouse anti- E. coli DnaK antibody (Enzo lifescience, Farmingdale, NY) by Western blot analysis.

Techniques: Inhibition, Binding Assay, Modification, Expressing

Journal: Gut Pathogens

Article Title: Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China

doi: 10.1186/s13099-023-00580-x

Figure Lengend Snippet: Maximum-likelihood phylogeny of vv16015 with other V. vulnificus. (vv16015 is marked with a red branch)

Article Snippet: 20 oyster samples were collected from the restaurant’s supplier and from which we isolated three strains of V. vulnificus : vv35, vv36, and vv37, which were sent with vv16015 to BGI (Shenzhen, China) for whole genome sequencing.

Techniques:

Journal: Gut Pathogens

Article Title: Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China

doi: 10.1186/s13099-023-00580-x

Figure Lengend Snippet: Kaplan-Meier survival curves of G. mellonella larvae infected with V. vulnificus strains at different inoculum sizes. (A) vv16015, (B) vv15018, and (C) vv220015. C1 = untreated control; C2 = saline control; T1 = 1 × 10 4 CFU/mL; T2 = 1 × 10 5 CFU/mL; T3 = 1 × 10 6 CFU/mL; T4 = 1 × 10 7 CFU/mL; T5 = 1 × 10 8 CFU/mL. (D) Number of surviving G. mellonella larvae after inoculation with different inoculum sizes

Article Snippet: 20 oyster samples were collected from the restaurant’s supplier and from which we isolated three strains of V. vulnificus : vv35, vv36, and vv37, which were sent with vv16015 to BGI (Shenzhen, China) for whole genome sequencing.

Techniques: Infection, Control, Saline

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: Expression of vvhBA upon exposure to murine blood and RAW 264.7 cells. A, the genes induced in V. vulnificus upon exposure to murine blood were identified by RNA-seq analysis. vvhBA was selected as the most highly induced extracellular toxin-encoding gene, and its induction was confirmed by qRT-PCR. Each column represents the vvhBA transcript level in V. vulnificus exposed to murine blood relative to M9G (negative control). Error bars represent the S.E. calculated using DeSeq2 for RNA-seq and the S.D. for qRT-PCR. B, V. vulnificus was exposed to DMEM (negative control) or RAW 264.7 cells in the presence or absence of l-NMMA. The vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript level in the cells exposed to DMEM without l-NMMA was set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: The effect of iscR mutation on vvhBA expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. **, p < 0.005; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311) and WT, a WT; ΔiscR (pJH0311) and ΔiscR, an iscR-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; iscR3CA, a strain expressing apo-locked IscR.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Mutagenesis, Expressing, Isolation, Quantitative RT-PCR, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: The effect of nitrosative stress and iron starvation on vvhBA and IscR expression. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3 and then exposed to 25 μm DEA NONOate for 20 min (A and B) or 50 μm DP for 10 min (C and D). A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT unexposed to DEA NONOate (A) or DP (C) were set to 1. Error bars represent the S.D. *, p < 0.05; ***, p < 0.0005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; ΔiscR, an iscR-deletion mutant.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: Specific binding of IscR to PvvhBA and sequences of the PvvhBA regulatory region. A, a 501-bp DNA of the PvvhBA regulatory region (5 nm) was radiolabeled and then incubated with increasing amounts of IscR as indicated. For competition analysis, various amounts of the unlabeled DNA fragment were used as a self-competitor and added to a reaction mixture containing 5 nm radiolabeled DNA and 30 nm IscR. B1, a DNA-IscR complex; F, free DNA. B, the same DNA of the PvvhBA regulatory region (32.3 nm) was labeled with 6-FAM, incubated with increasing amounts of IscR as indicated, and then digested with DNase I. The regions protected by IscR are indicated by black boxes (ISCRB1, ISCRB2, ISCRB3). Nucleotide numbers shown are relative to the transcription start site of vvhBA, which was determined previously (17). C, sequence analysis of the PvvhBA regulatory region. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid and dashed bent arrows, respectively. The putative −10 and −35 regions are underlined and the putative ribosome-binding site (AGGA) is boldface. The binding sequences of IscR are shown with the black boxes as described above. The binding sequences of HlyU (HLYUB; a white box) and H-NS (HNSB1, HNSB2, HNSB3, HNSB4, HNSB5, HNSB6; gray boxes) were determined later in this study (Fig. 6, C and D). The consensus sequences of the IscR-binding Type 2 DNA motif are indicated above the V. vulnificus DNA sequences. W, A or T; Y, C or T; R, A or G; x, any nucleotide.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Binding Assay, Incubation, Labeling, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: IscR and HlyU activate, but H-NS represses the vvhBA transcription. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05 relative to the WT; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT (pJH0311), a WT; ΔiscR (pJH0311), an iscR-deletion mutant; ΔhlyU (pJH0311), a hlyU-deletion mutant; Δhns (pJH0311), an hns-deletion mutant; ΔiscR (pJH0311::iscR), an iscR-complemented strain with pKK1531; ΔhlyU (pJH0311::hlyU), a hlyU-complemented strain with pZW1510; Δhns (pJH0311::hns), an hns-complemented strain with pGR1713.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: IscR relieves H-NS repression of vvhBA in cooperation with HlyU in vivo. Total RNA and proteins were isolated from the V. vulnificus strains grown aerobically to an A600 of 0.3. A and C, the vvhBA transcript levels were determined by qRT-PCR, and the vvhBA transcript levels in the WT were set to 1. Error bars represent the S.D. *, p < 0.05; **, p < 0.005; ns, not significant. B and D, the secreted VvhA and OmpU (internal control), and cellular IscR or IscR3CA, H-NS, HlyU, and DnaK (internal control) protein levels were determined by Western blot analysis. Molecular size markers (Bio-Rad) are shown in kDa. WT, a WT; Δhns, an hns-deletion mutant; ΔiscRΔhns, an iscR hns double-deletion mutant; iscR3CAΔhns, an hns-deletion mutant expressing apo-locked IscR; ΔiscR, an iscR-deletion mutant; ΔhlyU, a hlyU-deletion mutant; ΔiscRΔhlyU, an iscR hlyU double-deletion mutant; iscR3CAΔhlyU, a hlyU-deletion mutant expressing apo-locked IscR.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: In Vivo, Isolation, Quantitative RT-PCR, Western Blot, Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus

doi: 10.1074/jbc.RA120.012724

Figure Lengend Snippet: A proposed model for the regulation of vvhBA by multiple transcriptional regulators during host infection. Upon entering the host, V. vulnificus induces vvhBA expression in response to drastic environmental changes. IscR, along with HlyU, which is preferentially produced in the host (23), activates vvhBA by relieving H-NS repression by sensing nitrosative stress and iron starvation. Additionally, CRP activates vvhBA expression via Class I activation under certain nutrient-depleted conditions (17, 21). Meanwhile, a repressive interaction of H-NS and Fur at PvvhBA would be relieved in response to the increase in temperature and iron starvation in the host, respectively (32, 48). Taken together, the transcriptional regulators integrate diverse host-derived signals to collaboratively regulate vvhBA transcription during infection. Solid lines indicate activation of vvhBA by positive regulators, whereas dashed lines show relieved repression of vvhBA by negative regulators in the host. The transcription start site of vvhBA and the putative translational initiation codon of VvhB are indicated by solid bent arrows. The putative −10 and −35 regions, and ribosome-binding site (RBS) are underlined. ISCRB, an IscR-binding site; HLYUB, a HlyU-binding site; HNSB, an H-NS-binding site; CRPB, a CRP-binding site; FURB, a Fur-binding site.

Article Snippet: The purified IscR, HlyU, H-NS, OmpU, and truncated VvhA were used to raise rabbit polyclonal antibodies against the respective V. vulnificus proteins (AB Frontier, Seoul, South Korea).

Techniques: Infection, Expressing, Produced, Activation Assay, Derivative Assay, Binding Assay

Journal: Gut Pathogens

Article Title: Complete genome sequence of the Vibrio vulnificus strain VV2014DJH, a human-pathogenic bacterium isolated from a death case in China

doi: 10.1186/s13099-017-0216-7

Figure Lengend Snippet: Circular representations of the V. vulnificus strain VV2014DJH chromosomes. a Chromosome I and b chromosome II. From the outside to the center: the coding genes, gene function annotation results (COG, KEGG, GO), ncRNA genes, genomic G+C content, genome GC skew value distribution

Article Snippet: This whole genome sequences of V. vulnificus VV2014DJH has been deposited in the DDBJ/EMBL/GenBank database under the accession numbers CP019320 (ChI) and CP019321 (ChII).

Techniques:

Journal: Gut Pathogens

Article Title: Complete genome sequence of the Vibrio vulnificus strain VV2014DJH, a human-pathogenic bacterium isolated from a death case in China

doi: 10.1186/s13099-017-0216-7

Figure Lengend Snippet: General features of strain VV2014DJH genome

Article Snippet: This whole genome sequences of V. vulnificus VV2014DJH has been deposited in the DDBJ/EMBL/GenBank database under the accession numbers CP019320 (ChI) and CP019321 (ChII).

Techniques: