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The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways

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Sci. Signal.  16 May 2017:
Vol. 10, Issue 479, eaal4501
DOI: 10.1126/scisignal.aal4501
  • Fig. 1 V. para T3SS1 is cytotoxic to primary fibroblasts.

    (A) Schematic of T3SS1-induced cell death as previously defined in HeLa cells (14). The cell death is divided into stages defined by the action of known T3SS1 effectors (yellow, VopQ; cyan, VPA02450; magenta, VopS). (B) Representative confocal micrographs showing primary human fibroblasts infected with V. para POR3:T3SS1+. DNA is shown in blue (Hoechst), and actin is in green (phalloidin–Alexa Fluor 488). Scale bar, 20 μm. n = 3 independent experiments. (C) Lactate dehydrogenase (LDH) released by primary fibroblasts and HeLa cells infected with POR4:T3SS1 and POR3:T3SS1+. Percent cytotoxicity is relative to uninfected (UN) (negative) and Triton X-100–treated (positive) controls. Data represent the means of nine replicates [n = 3 (biological replicates) and 3 (technical replicates)] ± SD.

  • Fig. 2 V. para infection induces rapid changes in host gene expression.

    (A) Heat map of normalized differential expression between uninfected and V. para POR4:T3SS1-infected primary human fibroblasts over time. The transcripts depicted here met cutoffs of −1.5 ≥ FC ≥ 1.5, FDR ≤ 0.01, and log2CPM ≥ 0. Yellow indicates transcripts with higher abundance in POR4-infected fibroblasts compared to uninfected fibroblasts, and blue indicates transcripts with lower abundance in infected cells compared to uninfected cells. Clusters (colored bars on the left) were defined through hierarchical clustering of the differential expression data and represent groups of genes with similar temporal expression patterns. (B) Heat map of normalized differential expression between uninfected and POR3:T3SS1+-infected fibroblasts. Cutoffs and color coding are the same as in (A), and clusters were also assigned by hierarchical clustering.

  • Fig. 3 T3SS1 specifically induces the differential expression of 398 genes.

    (A) Heat map of normalized differential expression between primary human fibroblasts infected with V. para POR4:T3SS1 and those infected with V. para POR3:T3SS1+. The transcripts depicted here met cutoffs of −1.5 ≥ FC ≥ 1.5, FDR ≤ 0.01, and log2CPM ≥ 0 and were assigned into clusters by hierarchical clustering. Yellow denotes transcripts with increased abundance in POR3- compared to POR4-infected cells, and blue denotes a decreased abundance. (B) Heat map of log2FC values of the most significantly differentially expressed genes in (A). The log2FC between uninfected and either POR4- or POR3-infected fibroblasts is shown. Yellow indicates transcripts with increased abundance in infected cells compared to uninfected cells, and blue represents decreased abundance in infected cells compared to uninfected cells.

  • Fig. 4 V. para differentially affects core pathways based on the activity of T3SS1.

    Heat map of predicted activation (yellow) and repression (blue) Z scores calculated by Qiagen’s IPA using the temporal uninfected versus POR4:T3SS1 and uninfected versus POR3:T3SS1+ differential expression data. The color key correlates the displayed heat map color and calculated Z scores. Gray denotes that a pathway was unaffected (P < 0.05) in the infected condition relative to uninfected.

  • Fig. 5 T3SS1-specific induction of several transcripts requires MEK1/2 activity.

    (A) Immunoblot showing phosphorylated ERK1 and ERK2 (p-ERK1/2) and total ERK1/2 in POR3:T3SS1+-infected primary human fibroblasts treated with DMSO (vehicle) or U0126 to demonstrate the activity of U0126 as an MEK inhibitor in primary fibroblasts. n = 3 independent experiments. (B to E) qRT-PCR showing the expression of RHOB, JUN, FOS, PTGS2, EGR1, KLF2, and ATF3 in primary human fibroblasts that were pretreated with the MEK1/2 inhibitor U0126 or DMSO before being infected with POR3:T3SS1+ or POR4:T3SS1 compared to uninfected cells. Expression was normalized to the housekeeping gene IPO8. Data are 2−ΔΔCt ± SD. n = 3 experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way analysis of variance (ANOVA) analysis and Tukey’s multiple comparisons test. ns, not significant.

  • Fig. 6 Key differentially expressed genes activate cell survival in POR3-infected fibroblasts.

    Illustration of the action key POR3 differentially expressed genes in the cell survival network generated using IPA. The subcellular localization of the protein products of each gene and differential expression status (yellow, increased; blue, decreased) are depicted. Dashed lines denote indirect interactions. Orange lines indicate a gene expression state consistent with activation of cell survival; yellow lines indicate an expression state inconsistent with cell survival activation.

  • Fig. 7 V. para T3SS1 activates cell survival networks and represses cell death networks.

    (A) Heat map of IPA Z score prediction of activation (yellow) or repression (blue) of biological networks in response to POR3:T3SS1+ infection over time using the temporal uninfected versus POR3:T3SS1+ differential expression data from Fig. 2B. (B) Predicted cell survival and organismal death network activation (>0) and repression (<0) by POR3:T3SS1+ over the time course of T3SS1-mediated cell death. Plotted values correspond to the IPA Z scores from cell survival and organismal death networks in (A) and table S6.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/479/eaal4501/DC1

    Fig. S1. Confocal microscopy of POR4:T3SS1-infected fibroblasts.

    Fig. S2. Principal components analysis of RNA-seq data.

    Fig. S3. qRT-PCR analysis of cytokines in HeLa cells and RAW264.7 macrophages.

    Fig. S4. qRT-PCR analysis of top fibroblast POR4 versus POR3 differentially expressed genes in HeLa cells and RAW264.7 macrophages.

    Fig. S5. U0126 treatment inhibits POR3-induced Rhob expression in RAW264.7 macrophages.

    Table S1. Significantly differentially expressed genes in uninfected versus POR4:T3SS1-infected fibroblasts.

    Table S2. Significantly differentially expressed genes in uninfected versus POR3:T3SS1+-infected fibroblasts.

    Table S3. Significantly differentially expressed genes in POR4:T3SS1- versus POR3:T3SS1+-infected fibroblasts.

    Table S4. IPA canonical pathways uninfected versus POR4:T3SS1.

    Table S5. IPA canonical pathways uninfected versus POR3:T3SS1+.

    Table S6. IPA network analysis (disease and biological functions) of uninfected versus POR3:T3SS1+.

    Table S7. Primer list.

  • Supplementary Materials for:

    The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways

    Nicole J. De Nisco, Mohammed Kanchwala, Peng Li, Jessie Fernandez, Chao Xing, Kim Orth*

    *Corresponding author. Email: kim.orth{at}utsouthwestern.edu

    This PDF file includes:

    • Fig. S1. Confocal microscopy of POR4:T3SS1-infected fibroblasts.
    • Fig. S2. Principal components analysis of RNA-seq data.
    • Fig. S3. qRT-PCR analysis of cytokines in HeLa cells and RAW264.7 macrophages.
    • Fig. S4. qRT-PCR analysis of top fibroblast POR4 versus POR3 differentially expressed genes in HeLa cells and RAW264.7 macrophages.
    • Fig. S5. U0126 treatment inhibits POR3-induced Rhob expression in RAW264.7 macrophages.
    • Legends for tables S1 to S6
    • Table S7. Primer list.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 984 KB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Significantly differentially expressed genes in uninfected versus POR4:T3SS1-infected fibroblasts.
    • Table S2 (Microsoft Excel format). Significantly differentially expressed genes in uninfected versus POR3:T3SS1+-infected fibroblasts.
    • Table S3 (Microsoft Excel format). Significantly differentially expressed genes in POR4:T3SS1- versus POR3:T3SS1+-infected fibroblasts.
    • Table S4 (Microsoft Excel format). IPA canonical pathways uninfected versus POR4:T3SS1.
    • Table S5 (Microsoft Excel format). IPA canonical pathways uninfected versus POR3:T3SS1+.
    • Table S6 (Microsoft Excel format). IPA network analysis (disease and biological functions) of uninfected versus POR3:T3SS1+.

    [Download Tables S1 to S6]


    Citation: N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, K. Orth, The cytotoxic type 3 secretion system 1 of Vibrio rewires host gene expression to subvert cell death and activate cell survival pathways. Sci. Signal. 10, eaal4501 (2017).

    © 2017 American Association for the Advancement of Science