Research ArticleMicrobiology

Activation of master virulence regulator PhoP in acidic pH requires the Salmonella-specific protein UgtL

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Science Signaling  29 Aug 2017:
Vol. 10, Issue 494, eaan6284
DOI: 10.1126/scisignal.aan6284
  • Fig. 1 The ugtL gene is required for promoting the phosphorylated state of PhoP in acidic conditions in a PhoQ-dependent manner.

    (A) Phos-tag Western blot analysis of crude extracts prepared from WT S. enterica (JC805) and the isogenic ugtL mutant (JC925) strains grown in N-minimal media with 1 mM Mg2+ (pH 4.9; acidic pH), 10 μM Mg2+ (pH 7.6; low Mg2+), 1 mM Mg2+ (pH 7.6; noninducing), or the antimicrobial peptide C18G to mid-log phase using antibodies recognizing PhoP or the loading control AtpB. (B) Phos-tag Western blot analysis of crude extracts prepared from WT Salmonella (JC805) and ugtL mutant (JC925) harboring either the empty vector (pUHE) or a plasmid expressing UgtL (pUgtL) grown in inducing (acidic pH) or noninducing conditions to mid-log phase using antibodies recognizing PhoP or the loading control AtpB. (C) Abundance of mgtC, pcgL, pagC, ompC, and kdpE transcripts in WT (JC805) and ugtL (JC925) Salmonella grown in acidic pH to mid-log phase. The mean and SD from three independent experiments are shown. Unpaired Student’s t tests were performed between WT and isogenic ugtL mutant strains; **P < 0.01. (D) Phos-tag Western blot analysis of crude extracts prepared from WT (JC805), ugtL (JC925), phoP*phoQ (JC1014), and phoP*phoQ ugtL (JC1056) Salmonella grown in acidic pH to mid-log phase using antibodies directed against PhoP or the loading control AtpB. Data are representative of three independent experiments.

  • Fig. 2 The UgtL and PhoQ proteins interact.

    (A) β-galactosidase activity in bacterial two-hybrid system assays in E. coli BTH101 expressing the indicated fusion proteins. The bacteria carried the two B. pertussis adenylate cyclase fragments T25 and T18 either alone or fused to UgtL, CigR, or PhoQ in the indicated combinations. The adenylate cyclase fragment was fused to the N terminus in fusion proteins T25-UgtL, T25-CigR, and T18-PhoQ and to the C terminus in fusion protein PhoQ-T18. T25-Zip, Zip-T18, and T18-Zip were used as positive controls. The mean and SD from three independent experiments are shown. Unpaired Student’s t tests were performed between strains harboring empty vectors with the other combinations; *P < 0.05 and ****P < 0.0001. (B) Pull-down assays showing interactions between in vitro–synthesized UgtL-HA, PhoQ-FLAG, PhoZ-FLAG, and EnvZ-FLAG proteins. Samples were analyzed by Western blotting using antibodies recognizing the HA epitope and the FLAG epitope. Densitometry of each blot with ImageJ software is shown below the blots in the same order using arbitrary units (AU). Dashed lines in the densitometry graphs indicate signals from nonspecific binding of the UgtL-HA and FLAG-tagged proteins to antibodies recognizing the FLAG and HA epitopes, respectively. The data are representative of two independent experiments, which produced similar results.

  • Fig. 3 UgtL promotes autophosphorylation of PhoQ in vitro.

    (A) Amounts of PhoQ-P at the indicated time points in the presence or absence of UgtL and 32P-labeled ATP. (B) Amounts of PhoQ-P and PhoP-P at the indicated times after addition of PhoP to reaction mixtures containing PhoQ-P or PhoQ-P + UgtL. (C) Amounts of PhoP-P at the indicated times after addition of PhoP-P to reaction mixtures containing PhoQ or PhoQ + UgtL. The data are representative of two independent experiments, which produced similar results.

  • Fig. 4 UgtL and the cytoplasmic domain of PhoQ respond independently to mildly acidic pH.

    (A) Schematic of the full-length (FL) UgtL-FLAG protein and the truncated variants v1 to v6. AA, amino acid. (B) Phos-tag Western blot (top and middle) or Western blot (bottom) analysis of crude extracts prepared from Salmonella ugtL mutant (EG13682) harboring plasmids expressing FL FLAG-tagged UgtL or the indicated truncated variants. Phos-tag Western blots were probed with antibodies recognizing PhoP and AtpB (loading control), and the Western blot was probed with antibodies recognizing the FLAG epitope. Data are representative of three independent experiments, which produced similar results. (C) Phos-tag Western blot analysis of crude extracts prepared from WT Salmonella (JC805), ugtL-mutant Salmonella (JC925), Salmonella expressing a PhoQ variant (PhoQSB) that is insensitive to acidic pH (JC1102; phoQSB), and phoQSB ugtL Salmonella (JC1123) grown in acidic pH to mid-log phase. Blots were probed with antibodies recognizing PhoP and AtpB. Data are representative of three independent experiments, which produced similar results.

  • Fig. 5 UgtL is required for maximal Salmonella virulence in mice.

    Survival of BALB/c (A) or C3H/HeN (B) mice inoculated intraperitoneally with WT (14028s), ugtL (EG13682), or phoQ (MS5996s) Salmonella. Data are representative of n = 2 independent experiments, which produced similar results, n = 5 mice per each experimental group. Mantel-Cox test was performed between WT and ugtL Salmonella-infected mice; ****P < 0.0001.

  • Fig. 6 Activation of the sensor PhoQ in mildly acidic pH requires the Salmonella-specific UgtL protein to intensify the response of PhoQ to a mildly acidic pH.

    (A) The sensor PhoQ responds to acidification of the cytoplasm through its cytoplasmic domain. Mildly acidic pH causes PhoQ to autophosphorylate, after which it functions as a phosphodonor to the response regulator PhoP. Phosphorylated PhoP (PhoP-P) binds to promoters and promotes transcription of PhoP-activated genes. (B) UgtL enhances PhoQ autophosphorylation in response to acidic conditions, which results in increased abundance of PhoP-P and of PhoP-activated mRNAs. UgtL is required for Salmonella to achieve full PhoP-dependent gene transcription when the PhoQ-inducing signal is mildly acidic pH. (C) The periplasmic domain of PhoQ mediates PhoQ activation in response to conditions of low Mg2+ or the antimicrobial peptide C18G. In response to either of these stimuli, PhoQ undergoes autophosphorylation and then functions as a phosphodonor to PhoP. In this context, PhoQ-mediated activation of PhoP is sufficient for full transcription of PhoP-activated genes and is not dependent on UgtL.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/494/eaan6284/DC1

    Fig. S1. UgtL-dependent activation of the PhoP protein in mildly acidic pH is similar in WT and hns-FLAG Salmonella.

    Fig. S2. UgtL enhances PhoP-P abundance in an MgrB-independent manner.

    Fig. S3. UgtL enhances PhoP-P abundance posttranscriptionally.

    Fig. S4. The UgtL and PhoQ proteins interact.

    Fig. S5. FLAG-tagged UgtL functions as WT UgtL in vivo.

    Fig. S6. PhoP activates transcription of the ugtL gene in mildly acidic pH and low Mg2+ conditions.

    Fig. S7. UgtL promotes Salmonella resistance to magainin 2 in a PhoP-dependent manner.

    Table S1. Bacterial strains and plasmids used in this study.

    Table S2. Primers used in this study.

  • Supplementary Materials for:

    Activation of master virulence regulator PhoP in acidic pH requires the Salmonella-specific protein UgtL

    Jeongjoon Choi and Eduardo A. Groisman*

    *Corresponding author. Email: eduardo.groisman{at}yale.edu

    This PDF file includes:

    • Fig. S1. UgtL-dependent activation of the PhoP protein in mildly acidic pH is similar in WT and hns-FLAG Salmonella.
    • Fig. S2. UgtL enhances PhoP-P abundance in an MgrB-independent manner.
    • Fig. S3. UgtL enhances PhoP-P abundance posttranscriptionally.
    • Fig. S4. The UgtL and PhoQ proteins interact.
    • Fig. S5. FLAG-tagged UgtL functions as WT UgtL in vivo.
    • Fig. S6. PhoP activates transcription of the ugtL gene in mildly acidic pH and low Mg2+ conditions.
    • Fig. S7. UgtL promotes Salmonella resistance to magainin 2 in a PhoP-dependent manner.
    • Table S1. Bacterial strains and plasmids used in this study.
    • Table S2. Primers used in this study.

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    Citation: J. Choi, E. A. Groisman, Activation of master virulence regulator PhoP in acidic pH requires the Salmonella-specific protein UgtL. Sci. Signal. 10, eaan6284 (2017).

    © 2017 American Association for the Advancement of Science

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