Figures and data
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) The concentrations of upregulated amino acids in the mock- and Salmonella-infected groups (n = 4 biologically independent samples). (D) The concentrations of downregulated amino acids in the mock- and Salmonella-infected groups (n = 4 biologically independent samples). (E) Fold intracellular replication (20 h vs. 2 h) of Salmonella WT in RAW264.7 cells in the presence of 0.5, 1, 2, 4 mM β-alanine. Data are presented as the mean ± SD, n = 3 independent experiments. (F) Growth curves of Salmonella WT and the argT mutant (ΔargT) in N-minimal (left) and M9 minimal (right) medium supplemented with β-alanine (1 mM) as the sole carbon source. Data are presented as mean ± SD, n = 4 independent experiments. Statistical significance was assessed using two-sided Student’s t-test (C, D) and one-way ANOVA (E).
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) Fold intracellular replication (20 h vs. 2 h) of Salmonella Typhimurium 14028s wild type (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–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 immunofluorescence images and intracellular bacterial counts of Salmonella WT, ΔpanD, and cpanD in mouse liver at 5 days post-infection (green, Salmonella; blue, nuclei; scale bars, 50 µm). Images are representative of three independent experiments. The number of intracellular bacteria per infected cell was estimated in random fields, with n = 80 cells per group from 3 independent experiments. (F) 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-E). Statistical significance was assessed using the log-rank Mantel–Cox test (B), two-sided Student’s t-test (C), or one-way ANOVA (D, E).
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 partially 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. (E) The zinc levels in the livers of mice infection with either Salmonella WT or ΔpanD for 3 days, n = 5 mice per group. The data are presented as the mean ± SD (A–E). Statistical significance was assessed using one-way ANOVA (A–D), two-sided Student’s t-test (E). ns, not Significant.
β-Alanine promotes Salmonella replication within macrophages partially by increasing the expression of zinc transporter genes.
(A) The zinc levels in RAW264.7 cells after infection with Salmonella WT or ΔpanD for 8 h. (B) Replication of Salmonella WT, ΔpanD, ΔznuA and ΔpanDΔznuA in RAW264.7 cells. (C) Replication of Salmonella WT and ΔpanD in RAW264.7 cells in the presence or absence of 100 μM ZnSO4. (D) 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–D). Statistical significance was assessed using two-sided Student’s t-test (A), one-way ANOVA (B–D). ns, not Significant. (E) 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.
The levels of ROS and RNS in RAW264.7 cells after infection with Salmonella WT for 8 hours, in the presence or absence of 1 mM β-alanine.
Data are presented as mean ± SD, n = 3 independent experiments. Statistical significance was assessed using two-sided Student’s t-test. ns, not Significant.
Flow cytometry analysis was conducted to determine the percentage of pro-inflammatory M1 macrophages (CD86+) and anti-inflammatory M2 macrophages (CD163+).
RAW264.7 cells were infected with Salmonella WT for 8 hours, in the presence or absence of 1 mM β-alanine. Subsequently, the infected cells were collected for flow cytometry analysis. Representative dot plots and quantification of M1 (CD86+) and M2 (CD163+) macrophages are displayed in the left and right panels, respectively. Data are presented as mean ± SD, n = 3 independent experiments. Statistical significance was assessed using two-way ANOVA. ns, not Significant.
Growth curves of Salmonella WT and the cycA mutant (ΔcycA) in N-minimal medium supplemented with β-alanine (1 mM) as the sole carbon source.
Data are presented as mean ± SD, n = 3 independent experiments.
Growth curves of Salmonella WT and the cycA mutant (ΔcycA) in M9 minimal medium supplemented with β-alanine (1 mM) as the sole carbon source.
Data are presented as mean ± SD, n = 3 independent experiments.
Fold intracellular replication (20 h vs. 2 h) of Salmonella wild-type and ΔcycA in RAW264.7 cells.
Data are presented as mean ± SD, n = 4 independent experiments. Statistical significance was assessed using two-way ANOVA. ns, not Significant.
Liver and spleen bacterial burdens, and body weight of mice infected with Salmonella wild-type (STM) and cycA mutant (ΔcycA), at day 3 post-infection.
n = 5 mice per group. Statistical significance was assessed using Mann-Whitney U test. ns, not Significant.
Growth curves of Salmonella WT and the gabP mutant (ΔgabP) in N-minimal medium supplemented with β-alanine (1 mM) as the sole carbon source.
Data are presented as mean ± SD, n = 3 independent experiments.
Growth curves of Salmonella WT and the gabP mutant (ΔgabP) in M9 minimal medium supplemented with β-alanine (1 mM) as the sole carbon source.
Data are presented as mean ± SD, n = 3 independent experiments.
Growth curves of Salmonella WT, panD mutant (ΔpanD) and the complemented strain (cpanD) in LB medium.
Data are presented as mean ± SD, n = 4 independent experiments
Growth curves of Salmonella WT, panD mutant (ΔpanD) and the complemented strain (cpanD) in RPMI-1640 medium (B).
Data are presented as mean ± SD, n = 4 independent experiments.
Fold intracellular replication (20 h vs. 2 h) of Salmonella enterica serovar Typhi Ty2 wild type (WT), the panD mutant (ΔpanD) in human THP-1 monocyte like cell line (ATCC TIB-22).
Data are presented as mean ± SD, n = 4 independent experiments. Statistical significance was assessed using two-sided Student’s t-test. ns, not Significant.
Expression of SPI-2 is shown in the Z-score transformed heatmap, with orange representing higher and blue representing lower abundance.
Expression of SPI-1 is shown in the Z-score transformed heatmap, with orange representing higher and blue representing lower abundance.
Expression of SPI-3, SPI-4, and SPI-5 genes is shown in the Z-score transformed heatmap, with orange representing higher and blue representing lower abundance.
Strains and plasmids used in this study
