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

Abstract

Acidic conditions, such as those inside phagosomes, stimulate the intracellular pathogen Salmonella enterica to activate virulence genes. The sensor PhoQ responds to a mildly acidic pH by phosphorylating, and thereby activating, the virulence regulator PhoP. This PhoP/PhoQ two-component system is conserved in a subset of Gram-negative bacteria. PhoQ is thought to be sufficient to activate PhoP in mildly acidic pH. However, we found that the Salmonella-specific protein UgtL, which was horizontally acquired by Salmonella before the divergence of S. enterica and Salmonella bongori, was also necessary for PhoQ to activate PhoP under mildly acidic pH conditions but not for PhoQ to activate PhoP in response to low Mg²⁺ or the antimicrobial peptide C18G. UgtL increased the abundance of phosphorylated PhoP by stimulating autophosphorylation of PhoQ, thereby increasing the amount of the phosphodonor for PhoP. Deletion of ugtL attenuated Salmonella virulence and further reduced PhoP activation in a strain bearing a form of PhoQ that is not responsive to acidic pH. These data suggest that when Salmonella experiences mildly acidic pH, PhoP activation requires PhoQ to detect pH and UgtL to amplify the PhoQ response. Our findings reveal how acquisition of a foreign gene can strengthen signal responsiveness in an ancestral regulatory system.

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 wild-type (WT) Salmonella enterica (JC805) and the isogenic ugtL mutant (JC925) strains grown in N-minimal media with 1 mM of Mg²⁺ at pH 4.9 (acidic pH), 10 μM Mg²⁺ at pH 7.6 (low Mg²⁺), 1mM Mg²⁺ at pH 7.6 (non-inducing), 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 wild-type Salmonella (JC805) and ugtL mutants (JC925) harboring either the empty vector (pUHE) or a plasmid expressing UgtL (pUgtL) grown in inducing conditions (acidic pH) or non-inducing 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 wild-type (JC805) and ugtL (JC925) Salmonella grown in acidic pH to mid-log phase. The mean and SD from three independent experiments are shown. Unpaired students T-test were performed between wild-type and isogenic ugtL mutant strains; ** p < 0.01. (D) Phos-tag Western blot analysis of crude extracts prepared from wild-type (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. In each experimental condition, the bacteria carried the two Bordetella 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 students T-test were performed between strains harboring empty vectors with the other combinations; * p < 0.05, **** p <0.0001. (B) Pulldown 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–v6. (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 truncated variants as indicated. Phos-tag Western blots were probed with antibodies recognizing PhoP and AtpB (loading control), and the Western blot was probled 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 wild-type (WT) Salmonella (JC805), ugtL mutant Salmonella (JC925), Salmonella expressing a PhoQ variant (PhoQ^SB) that is insensitive to acidic pH (JC1102; phoQ^SB), and phoQ^SB 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 wild-type (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 wild-type 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.