Source: https://iai.asm.org/content/86/9/e00932-17/figures-only
Timestamp: 2019-04-22 12:23:51+00:00

Document:
Effect of cFP on survival of V. vulnificus under H2O2-induced oxidative stress conditions. (A) Culture supernatant from wild-type V. vulnificus cells at stationary phase contributes to survival of V. vulnificus under oxidative stress. Wild-type V. vulnificus cells were treated with an ethyl acetate extract of supernatant from wild-type V. vulnificus or ΔllcA isotype cultures grown in LB broth to exponential phase (A600 = 0.1) or stationary phase (A600 = 2.5), and 625 μM H2O2 was added to each sample. Survival of cells was assessed by measuring CFU. (B) cFP at 1 mM increases survivability of V. vulnificus under H2O2-induced oxidative stress. Wild-type V. vulnificus cells grown in the presence of different concentrations of cFP were treated with 625 μM H2O2 (solid bars) or no H2O2 treatment (open bars). Cell survival was then assessed by measuring CFU. CFU were expressed as the CFU60/CFU0 ratio. CFU0 and CFU60 are CFU per milliliter at 0 min and at 60 min, respectively, after treatment or not with H2O2. The error bars denote standard deviations of the results of three independent experiments (**, P < 0.005; *, P < 0.05; NS, not significant).
Expression of katG is influenced by cFP and rpoS. (A) Transcription levels of katG as measured by β-galactosidase activities from wild-type V. vulnificus MO6-24/O [Wild type (pRK415)]; an isotype, KPR101, with rpoS deleted [ΔrpoS (pRK415)]; and KPR101 complemented with the rpoS gene [ΔrpoS (pRK-rpoS)] harboring pMZtc-katG at 0 mM, 1 mM, and 5 mM cFP under H2O2-induced oxidative conditions or without H2O2. H2O2 (625 μM) was added during the exponential growth phase of the cells (A600, ∼0.1) in AB broth. The data are the average values of the results of three independent experiments, and the error bars denote standard deviations. MU, Miller units. (B) Effect of 1 mM cFP on the survival of V. vulnificus MO6-24/O (Wild type) and KPR101 (ΔrpoS) strains in the presence of 625 μM H2O2. The error bars denote standard deviations of three independent experiments (**, P < 0.005; *, P < 0.05; NS, not significant).
RpoS expression is enhanced at the posttranscriptional level in the presence of 1 mM cFP. (A) Transcription levels of rpoS as measured by β-galactosidase activities (solid lines) and growth curves (dashed lines) of V. vulnificus MO6-24/O cultured in AB broth harboring pMZtc-rpoS (rpoS-lacZ fusion) and treated with 0 mM, 1 mM, and 5 mM cFP. The data are the averages of the results of three independent experiments, and the error bars denote standard deviations. M.U., Miller units. (B) Western hybridization using polyclonal antisera against RpoS of total protein extracts from V. vulnificus MO6-24/O cultured in AB broth treated in early stationary phase with cFP at 0 mM, 1 mM, and 5 mM. (From left) Lane 1, treated with DMSO (dimethyl sulfoxide) (no cFP); lane 2, treated with 1 mM cFP; lane 3, treated with 5 mM cFP; 40 μg of the total protein from each sample was loaded on the gel. For a loading control, antiserum against SidC (insulin-degrading enzyme) (59), a protein that is not modulated by cFP, was used. The relative intensities of the bands were measured using Multi Gauge ver. 3.0 (Fujifilm, Tokyo, Japan).
RpoS expression is regulated by cFP via vHUαβ. (A) The effect of cFP on the expression levels of clpX, rssB, dksA, hfq, hns, and vhuAB was assessed by qRT-PCR. RNA was isolated from wild-type V. vulnificus MO6-24/O cultured in AB broth and treated with 0 mM, 1 mM cFP, and 5 mM cFP at early stationary phase (A600, ∼0.2). (B) Expression levels of RpoS as measured by qRT-PCR. RNA samples were obtained from wild-type (pRK415), ΔvhuAB(pRK415), and ΔvhuAB(pRK-vhuAB) cultured in AB broth at early stationary phase (A600, ∼0.2). All the RNA levels were quantified using the comparative threshold cycle (ΔΔCT) method, and RNA fold change values were normalized to the value for MO6-24/O without cFP (*, P < 0.05; NS, not significant).
cFP enhances the expression of genes encoding subunits of a histone-like protein, vHUα and vHUβ, via LeuO. (A) Expression levels of rpoS as measured by qRT-PCR from wild-type V. vulnificus [Wild type (pBBR1-MCS2)], ΔleuO(pBBR1-MCS2), ΔleuO(pBBR1-leuO), ΔleuOΔvhuAB(pRK415), and ΔleuOΔvhuAB(pRK-vhuAB) cultured in AB broth at early stationary phase (A600, ∼0.2). (B) β-Galactosidase activities from wild-type (pBBR1-MCS2), ΔleuO(pBBR1-MCS2), and ΔleuO(pBBR1-leuO) strains harboring pMZtc-vhuA (left) or pMZtc-vhuB (right). Overnight cultures of V. vulnificus were subcultured into fresh LB broth supplemented with each concentration of cFP, and when the cells reached exponential phase (optical density [OD], 0.3 to 0.4), the cells were resubcultured in fresh AB broth. β-Galactosidase activities were measured as described in Materials and Methods. The error bars denote standard deviations of the results of three independent experiments. MU, Miller units. **, P < 0.005; *, P < 0.05; NS, not significant. (C) Binding of recombinant LeuO (rLeuO) to the upstream regions of vhuA (left) and vhuB (right) genes as determined by electrophoretic mobility shift assay. Ten nanograms of radiolabeled probes was incubated with increasing concentrations of LeuO. Lanes 1 to 5, LeuO concentrations of 0 nM, 10 nM, 20 nM, 40 nM, and 80 nM, respectively; lanes 6 to 8, 80 nM rLeuO with unlabeled probes as a competitor at 1 ng, 10 ng, and 100 ng, respectively.
Transcriptional levels of leuO under various concentrations of cFP and transcriptional levels of vhuA and vhuB modulated by LeuO differentially expressed in the arabinose induction system. (A) Transcription levels of LeuO as measured by β-galactosidase activities of V. vulnificus MO6-24/O harboring pMZtc-leuO cultured in AB broth and treated with 0 mM, 1 mM, or 5 mM cFP. (B) Transcription levels of vhuA (left) and vhuB (right) as measured by β-galactosidase activities from MO6-24/O(pBBR1-MCS2) harboring pMZtc-vhuA or pMZtc-vhuB and from ΔleuO(pBBR12-leuO-ara) harboring pMZtc-vhuA or pMZtc-vhuB cultured in AB broth. The culture conditions are described in Materials and Methods. To induce the expression of LeuO by arabinose, the culture was split into seven aliquots when the A600 reached 0.1, and then various concentrations of arabinose (0, 0.005, 0.01, 0.05, 0.1, 0.5, and 1%) were added. The error bars denote standard deviations of the results of three independent experiments. MU, Miller units.
Enhancement of stability of RpoS mRNA by cFP. (A) The relative mRNA level of rpoS after rifampin treatment was measured by qRT-PCR. Wild-type V. vulnificus MO6-24/O and the ΔvhuAB deletion mutant were treated with rifampin when growth in AB broth reached an A600 of 0.2. At 0, 5, 10, 20, and 40 min after the treatment, RNA was isolated from cells for qRT-PCR analysis. Half-lives were calculated using GraphPad Prism 5. The data are average values from three independent samples, and the error bars denote standard deviations. (B) Translation level of rpoS as measured by β-galactosidase activity and Western hybridization using antiserum against RpoS of V. vulnificus wild type (pBBR1-MCS2), ΔvhuAB(pBBR1-MCS2), and ΔvhuAB(pBBR12-vhuAB) harboring pRZtl-rpoS cultured in AB broth. Measurements of β-galactosidase activities and Western hybridization were performed as described in Materials and Methods. The error bars denote standard deviations of the results of three independent experiments. MU, Miller units. **, P < 0.005; *, P < 0.05; NS, not significant).
Transcription of the RpoS-inducing genes aldA, gabD, and vvpE is also induced by 1 mM cFP. A comparison of the transcription levels of aldA (A), gabD (B), and vvpE (C) in wild-type V. vulnificus MO6-24/O and KPR101 (ΔrpoS) cultured in AB broth is shown. Cells were treated with cFP (0, 1, or 5 mM), and RNA samples obtained at early stationary phase (A600, ∼0.2) were analyzed by qRT-PCR using the primers shown in Table S2 in the supplemental material. Overnight cultures were subcultured in AB minimal media supplemented with cFP (0, 1, and 5 mM). RNA levels were quantified using the ΔΔCT method, and the RNA fold change was normalized to the value for MO6-24/O cultured with 0 mM cFP (DMSO buffer only). The data are average values from three independent samples, and the error bars denote the standard deviations (***, P < 0.001; **, P < 0.005; *, P < 0.05; NS, not significant).
Effect of cFP on expression of genes encoding histone-like protein HUα and HUβ subunits, RpoS, and LeuO in V. cholerae and V. parahaemolyticus. Shown is a comparison of transcription levels of vhuA, vhuB, rpoS, and leuO treated with cFP in wild-type V. cholerae (A) and V. parahaemolyticus (B) at early stationary phase (A600, ∼0.2) by qRT-PCR using the primers shown in Table S2 in the supplemental material. Overnight cultures were subcultured in LB medium and then subcultured in AB minimal media containing cFP (0, 0.5, 1, and 5 mM). RNA levels were quantified using the ΔΔCT method, and the RNA fold change of each gene was normalized to the value for cells cultured with 0 mM cFP (DMSO only). The data are average values from three independent samples, and the error bars denote standard deviations (***, P < 0.001; **, P < 0.005; *, P < 0.05; NS, not significant).
Role of cFP as a virulence factor while in the host and also as a signal to induce expression of genes to protect the pathogen. cFP produced by V. vulnificus suppresses the immune response of human cells by inhibiting nuclear translocation of NF-κB (32). It also suppresses regulators responsible for the expression of ROS scavengers, resulting in higher intracellular ROS levels in human cells and ultimately leading to apoptosis (31). The enhanced ROS production in human cells could cause damage to the cognate pathogen. However, cFP also acts as a signal to trigger a signal transduction pathway in the pathogen composed of ToxR-LeuO-vHUαβ. This signaling cascade stabilizes the mRNA of the alternate sigma factor RpoS. RpoS induces transcription of katG, encoding a peroxidase that detoxifies ROS, thereby protecting the pathogen. cFP signaling also controls the ToxR, LeuO, vHUαβ, and RpoS regulons, leading to modified regulation of numerous genes, which could be responsible for V. vulnificus pathogenesis; i.m., inner membrane; o.m., outer membrane.
Table S1. Bacterial strains and plasmids used in this study. Table S2. Primers used in this study. Fig. S1. cFP does not have scavenging activity of DPPH radical.

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