Acetylation of PhoP K88 Is Involved in Regulating Salmonella Virulence

Abstract

The PhoP-PhoQ two-component regulation system of Salmonella enterica serovar Typhimurium is involved in the response to various environmental stresses and is essential for bacterial virulence. Our previous studies showed that acetylation plays an important role in regulating the activity of PhoP, which consequently mediates the change in virulence of S Typhimurium. Here, we demonstrate that a conserved lysine residue, K88, is crucial for the function of PhoP and its acetylation-downregulated PhoP activities. K88 could be specifically acetylated by acetyl phosphate (AcP), and the acetylation level of K88 decreased significantly after phagocytosis of S Typhimurium by macrophages. Acetylation of K88 inhibited PhoP dimerization and DNA-binding abilities. In addition, mutation of K88 to glutamine, mimicking the acetylated form, dramatically attenuated intestinal inflammation and systemic infection of S Typhimurium in the mouse model. These findings indicate that nonenzymatic acetylation of PhoP by AcP is a fine-tuned mechanism for the virulence of S Typhimurium and highlights that virulence and metabolism in the host are closely linked.

Keywords: PhoP; Salmonella enterica serovar Typhimurium; acetyl phosphate; acetylation; virulence.

FIG 1

Acetylation of K88 inhibits the activity of PhoP. (A) His-tagged PhoP was purified and analyzed with LC-MS-MS after trypsin digestion. Shown is the MS-MS spectrum of the peptide E₈₃GWQDKVEVLSSGADDYVTKPFHIEEVMAR₁₁₂. The presence of a series of b ions supports the acetylation of K88 in the peptide with the key fragment ions in red. (B) The asterisk denotes the conserved K88 residue. The result was analyzed with BioEdit 7.0. (C) The proliferation of bacteria in RAW264.7 cells. RAW264.7 cells were infected by the wild-type strain (eWT) or phoP K88 derivative mutant strains. Infected cells were lysed at 2 h or 24 h postinfection. The number of viable intracellular bacteria was determined. Bacterial growth was measured as the fold change in CFU between 2 h and 24 h. Results are shown as the mean ± SD from three independent experiments; ****, P < 0.0001. Student’s t test. (D) The proliferation of bacteria in primary peritoneal macrophages. After the wild-type (eWT) and phoP K88 derivative mutant strains infected primary peritoneal macrophages for 2 h and 24 h, respectively, viable cells were counted at the indicated time point. The growth fold change between 2 h and 24 h was calculated. Results are shown as the mean ± SD from three independent experiments; **, P < 0.01. Student’s t test. (E) The transcriptional levels of phoP- and phoP-regulated genes in the wild-type strain (eWT) and phoP K88 mutants. Total RNA was harvested from bacteria grown to an OD₆₀₀ of ∼0.4 in M9CA medium with 8 µM magnesium. The mRNA levels were determined by qPCR. The relative expression of genes in eWT was set as 1. Error bars indicate ± SDs of triplicate measurements; **, P < 0.01; ***, P < 0.001. Student’s t test. (F) Invasion efficiency of bacteria in HeLa cells. After the wild-type (eWT) and phoP K88 derivative mutant strains infected HeLa cells for 2 h, viable bacteria were counted by plating on agar plates to calculate invasion efficiency. Error bars represents the ± SD from three independent experiments; ****, P < 0.0001. Student’s t test.

FIG 2

K88 can be acetylated by AcP. (A) The specificity of anti-K88Ac antibody. Wild-type PhoP protein (WT) and site-specific acetylated K88Ac, K102Ac, and K201Ac proteins were resolved on 12% SDS-PAGE and probed with anti-K88Ac antibody and anti-PhoP antibody. (B) The acetylation level of PhoP K88 in macrophages. After infecting RAW264.7 cells for 24 h, the intracellular bacteria were harvested for IP assay. The immunoprecipitated PhoP protein by anti-Flag antibody was analyzed by Western blotting with anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments; **, P < 0.01. Student’s t test. (C) The acetylation of PhoP K88 after knockout of pat or cobB. PhoP proteins from the wild-type strain and mutation strain of S. Typhimurium 14208s were detected by Western blotting and probed with anti-K88Ac antibody, and anti-PhoP antibody was used as a loading control. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments. *, P < 0.05. ns, no significance. Student’s t test. (D) The acetylation level of PhoP K88 isolated from strains cultured in LB medium supplemented with glucose. The wild-type strain and pta or ackA mutation strains of S. Typhimurium 14208s were cultured in LB medium with or without 0.8% (wt/vol) glucose added and then harvested for IP assay. The immunoprecipitated PhoP protein by anti-Flag antibody was analyzed by Western blotting with anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative K88 acetylation level over PhoP was quantified (top panel). Error bars indicate ± SDs of triplicate experiments; *, P < 0.05. Student’s t test. (E) PhoP K88 was acetylated by AcP in vitro. Purified His-tagged PhoP was incubated with AcP as an acetyl group donor for different durations, and then samples were detected by Western blot analysis with anti-pan acetyl lysine antibody (α-Pan Ac), which recognized acetylated lysine residues and anti-K88Ac antibody. Representative results from three independent experiments of Western blots are shown. The relative pan acetylation or K88 acetylation level over PhoP was quantified. Error bars indicate ± SDs of triplicate experiments; *, P < 0.05; **, P < 0.01. ns, no significance. Student’s t test.

FIG 3

Acetylation of K88 impairs the dimer formation and DNA-binding ability of PhoP. (A) The phosphorylation of PhoP K88Ac in vitro. Purified wild-type PhoP and PhoP K88Ac proteins were incubated with or without 20 mM PAM, respectively. The samples were resolved on 12% SDS-PAGE gel containing Phos-tag and analyzed with anti-His antibody. Representative results from three independent experiments of Western blots are shown. (B) Dimer-formation of PhoP K88Ac. Purified wild-type PhoP or K88Ac was subjected to cross-linking with DSS. Western blots were probed with anti-His antibody and independently repeated at least three times. The relative ratio of dimer over monomer of PhoP was quantified. Error bars indicate ± SDs of triplicate experiments; **, P < 0.01. Student’s t test. (C) DNA-binding activity of PhoP K88Ac assessed by EMSA. 6′-FAM-labeled phoP promoter was incubated with His-tagged wild-type PhoP or PhoP K88Ac at the indicated concentration, and then the reaction products were analyzed in 5% PAGE. Representative EMSA results from three independent experiments are shown. (D) DNA-binding activity of PhoP K88 derivative mutants compared to WT in log-phase acid tolerance response by ChIP. eWT or phoP K88 mutant strains in which phoP was flag-tagged in chromosome were cultured in EG medium at pH 5.0 for 1 h and harvested. The total cell lysate was immunoprecipitated by anti-Flag antibody. The candidate gene promoter before (input) and after (Flag-ChIP) were quantified by qPCR and represented as the relative ratio of ChIP/input. Results are shown as mean ± SD from three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, no significance. Student’s t test.

FIG 4

Acetylation of PhoP K88 modulates bacterial virulence in the mouse model. (A) Survival rates of mice infected by oral gavage. BALB/c mice were infected by 1.5 × 10⁷ CFU of bacteria (wild-type or phoP K88 mutant strains) or PBS as the control through oral gavage. The number of live mice was counted twice a day. (B) Survival rates of mice infected by intraperitoneal injection. BALB/c mice were injected intraperitoneally with 1.5 × 10⁵ CFU of bacteria (eWT or phoP K88 mutant strains) or PBS as the control. The number of live mice was counted twice a day. (C) Bacterial burdens in liver and spleen. The livers and spleens were harvested 48 h after oral infection. The amounts of bacteria in the liver and spleen were counted. Results are shown as the mean ± SD; **, P < 0.01; ***, P < 0.001. ns, no significance. Student’s t test. (D) Immunofluorescence staining of S. Typhimurium cecal colonization in vivo. The ceca from streptomycin-treated BALB/c mice after oral infection were prepared as paraformaldehyde-fixed paraffin section. These sections were stained for S. Typhimurium lipopolysaccharide (LPS) (red), actin (green), and nuclei with DAPI (blue). Images are pseudocolor representations at ×200 magnification. (E) H&E-stained ceca of mice. The ceca were fixed and embedded in paraffin to slice a section which was stained with H&E. L, intestinal lumen; e, edema; sa, submucosa. (F) Neutrophil infiltration in ceca. The paraffin section was stained with hematoxylin and anti-MPO antibody by immunohistochemistry. Claybank indicates PMN and blue indicates the nucleus.