Host-derived β-alanine promotes Salmonella replication inside macrophages.

(A) Schematic workflow for targeted metabolomics investigation of mock- and Salmonella-infected (STM) mouse RAW264.7 macrophages. Picture materials were used from bioicons (https://bioicons.com/). (B) Principal component analysis (PCA) score plots of metabolic profiles in the mock- and Salmonella-infected (STM) groups (n = 4 biologically independent samples). (C) Relative copiousness of amino acids that were differentially abundant in the Salmonella-infected groups versus the mock groups. (D) The concentrations of β-alanine in the mock- and Salmonella-infected groups. (E) Relative fold replication of Salmonella WT in RAW264.7 cells in the presence or absence of 1 mM β-alanine. (F) qRT‒PCR analysis of the expression of pro- (Il1b, Tnf) and anti-inflammatory genes (Il10, Il4ra) of RAW264.7 cells after infection with Salmonella WT for 8 h, in the presence or absence of 1 mM β-alanine. (G) Flow cytometry analysis of percentage of pro-inflammatory M1 macrophages (CD86+) and anti-inflammatory M2 macrophages (CD163+). RAW264.7 cell were infected Salmonella WT for 8 h, in the presence or absence of 1 mM β-alanine, and then the infected cells were collected for flow cytometry analysis. Representative dot plot and quantification of M1 (CD86+) or M2 (CD163+) macrophages are shown in the left and right panel, respectively. The data are presented as the mean ± SD, n = 3 independent experiments (E-G). Statistical significance was assessed using two-sided Student’s t-test (D, E) and two-way ANOVA (F, G)

De novo β-alanine synthesis is critical for Salmonella replication inside macrophages.

(A) Scheme of β-alanine and the downstream CoA biosynthesis pathway in Salmonella. (B) qRT‒PCR analysis of the expression of the Salmonella panD gene in RAW264.7 cells (8 h postinfection) and RPMI-1640 medium. (C) qRT‒ PCR analysis of the expression of the Salmonella panD gene in N-minimal medium and LB medium. (D) Expression of the panD-lux transcriptional fusion in N-minimal medium and LB medium. Luminescence values were normalized to 105 bacterial CFUs. (E) Relative fold replication of Salmonella WT, the panD mutant (ΔpanD) and the complemented strain (cpanD) in RAW264.7 cells. (F) Number of intracellular Salmonella WT, ΔpanD, and cpanD strains per RAW264.7 cell at 2 and 20 h postinfection. The number of intracellular bacteria per infected cell was estimated in random fields, n = 80 cells per group from 3 independent experiments. (G) Representative immunofluorescence images of Salmonella WT, ΔpanD, and cpanD in RAW264.7 cells at 20 h postinfection (green, Salmonella; blue, nuclei; scale bars, 50 µm). Images are representative of three independent experiments. (H) Replication of Salmonella WT and ΔpanD in RAW264.7 cells in the presence or absence of 1 mM β-alanine. The data are presented as the mean ± SD, n = 3 (B–D) or n =4 (E, H) independent experiments. Statistical significance was assessed using two-sided Student’s t-test (B-D) or one-way ANOVA (E, F, H). ns, not Significant.

De novo β-alanine synthesis is critical for systemic Salmonella infection in mice.

(A) Schematic illustration of the mouse infection assays. Picture materials were used from bioicons (https://bioicons.com/). (B, C) Survival curves (B) and body weight dynamics (C) of mice infected i.p. with Salmonella WT, ΔpanD, or cpanD. n = 10 mice per group. (D) Liver and spleen bacterial burdens and body weights of mice infected with Salmonella WT, ΔpanD, or cpanD on day 3 postinfection. n = 7 mice per group. (E) Representative H&E-stained liver sections from mice that were left uninfected or infected with Salmonella WT, ΔpanD, or cpanD on day 5 postinfection. Arrows indicate severe inflammatory cell infiltration in the mouse liver. Images are representative of three independent experiments. The data are presented as the mean ± SD (B-D). Statistical significance was assessed using the log-rank Mantel–Cox test (B), two-sided Student’s t-test (C), or one-way ANOVA (D).

β-Alanine is involved in the regulation of several metabolic pathways in Salmonella.

(A) Principal component analysis (PCA) score plots of transcriptomic profiles of Salmonella WT and ΔpanD (n = 3 biologically independent samples). (B) Volcano plot of the differentially expressed genes (DEGs) in Salmonella WT versus ΔpanD. The upper right section (red dots) indicates the upregulated DEGs, and the upper left section (green dots) indicates the downregulated DEGs. (C) Gene Ontology (GO) enrichment analysis of DEGs. Bubble chart showing the top 20 enriched GO terms. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. (E) Expression of the downregulated pathways (activated by PanD) is shown in the Z score-transformed heatmap, with red representing higher abundance and blue representing lower abundance. (F) qRT‒PCR analysis of the mRNA levels of 16 selected downregulated DEGs in Salmonella WT, ΔpanD, and cpanD. The data are presented as the mean ± SD, n = 3 independent experiments. Statistical significance was assessed using two-way ANOVA.

β-Alanine promotes Salmonella virulence in vivo by increasing the expression of zinc transporter genes.

(A, B, C) Liver (A) and spleen (B) bacterial burdens and body weight (C) of mice infected with Salmonella WT, ΔfadAB, ΔmetR, ΔhisABCDFGHL, ΔkdpABC, ΔmglABC, ΔpotFGHI, or ΔleuO on day 3 postinfection. n = 5 mice per group. (D) Liver and spleen bacterial burdens and body weights of mice infected with Salmonella WT, ΔpanD, ΔznuA or ΔpanDΔznuA on day 3 postinfection. n = 5 mice per group. The data are presented as the mean ± SD (A–D). Statistical significance was assessed using one-way ANOVA (A–D). ns, not Significant.

β-Alanine promotes Salmonella replication within macrophages by increasing the expression of zinc transporter genes.

(A) Replication of Salmonella WT, ΔpanD, ΔznuA and ΔpanDΔznuA in RAW264.7 cells. (B) Replication of Salmonella WT and ΔpanD in RAW264.7 cells in the presence or absence of 100 μM ZnSO4. (C) Replication of Salmonella WT and ΔznuA in RAW264.7 cells in the presence or absence of 1 mM β-alanine. The data are presented as the mean ± SD, n = 3 independent experiments (A–C). Statistical significance was assessed using one-way ANOVA (A–C). ns, not Significant. (D) Schematic model of β-alanine-mediated Salmonella replication inside macrophages. In macrophages, Salmonella acquires β-alanine both via the uptake of β-alanine from host macrophages and the de novo synthesis of β-alanine. β-alanine promotes the expression of zinc transporter genes ZnuABC, which facilitate the uptake of zinc by intracellular Salmonella, therefore promote Salmonella replication in macrophages and subsequent systemic infection.