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. 2025 Jun 19:13:RP103714.
doi: 10.7554/eLife.103714.

Salmonella exploits host- and bacterial-derived β-alanine for replication inside host macrophages

Shuai Ma #  1 , Bin Yang #  1 , Yuyang Sun  1 , Xinyue Wang  1 , Houliang Guo  1 , Ruiying Liu  1 , Ting Ye  1 , Chenbo Kang  1 , Jingnan Chen  1 , Lingyan Jiang  1
Affiliations

Salmonella exploits host- and bacterial-derived β-alanine for replication inside host macrophages

Shuai Ma et al. Elife. .

Abstract

Salmonella is a major foodborne pathogen that can effectively replicate inside host macrophages to establish life-threatening systemic infections. Salmonella must utilize diverse nutrients for growth in nutrient-poor macrophages, but which nutrients are required for intracellular Salmonella growth is largely unknown. Here, we found that either acquisition from the host or de novo synthesis of a nonprotein amino acid, β-alanine, is critical for Salmonella replication inside macrophages. The concentration of β-alanine is decreased in Salmonella-infected macrophages, while the addition of exogenous β-alanine enhances Salmonella replication in macrophages, suggesting that Salmonella can uptake host-derived β-alanine for intracellular growth. Moreover, the expression of panD, the rate-limiting gene required for β-alanine synthesis in Salmonella, is upregulated when Salmonella enters macrophages. Mutation of panD impaired Salmonella replication in macrophages and colonization in the mouse liver and spleen, indicating that de novo synthesis of β-alanine is essential for intracellular Salmonella growth and systemic infection. Additionally, we revealed that β-alanine influences Salmonella intracellular replication and in vivo virulence partially by increasing expression of the zinc transporter genes znuABC, which in turn facilitates the uptake of the essential micronutrient zinc by Salmonella. Taken together, these findings highlight the important role of β-alanine in the intracellular replication and virulence of Salmonella, and panD is a promising target for controlling systemic Salmonella infection.

Keywords: Salmonella; infectious disease; microbiology; replication in macrophage; virulence; zinc uptake; β-alanine.

Plain language summary

Salmonella, a type of bacterium, is one of the most common foodborne pathogens and is responsible for illnesses ranging from gastroenteritis to typhoid fever. Each year, it infects over 100 million people globally, leading to 350,000 deaths. Immune cells called macrophages are important for defending against bacterial infections as they can engulf and destroy harmful bacteria. However, Salmonella is able to survive and multiply within these very immune cells that are meant to eliminate it. To do so, the bacteria require nutrients such as amino acids, which are the building blocks of proteins. These are either produced by the bacteria or obtained from the infected host. However, the specific nutrients that Salmonella require to survive and multiply, as well as their source, remained unknown. To investigate, Ma, Yang et al. measured amino acid levels in macrophages that had been infected with Salmonella and compared them to those in uninfected macrophages. This revealed that the levels of an amino acid called β-alanine – which differs from many amino acids because it is not used to make proteins – are lower in infected macrophages. Furthermore, providing infected macrophages with more β-alanine increased bacterial replication. This suggests that the bacteria acquire this amino acid from the macrophages in order to survive and replicate. To determine whether Salmonella can also make β-alanine themselves, Ma et al. prevented them from producing it, which slowed bacterial growth and led to milder infections in mice. This ability to produce β-alanine required a gene known as PanD. Further experiments also showed that β-alanine assists Salmonella in acquiring the essential micronutrient zinc from macrophages. Taken together, the findings of Ma et al. reveal the critical role of β-alanine in Salmonella growth within macrophages and its ability to cause disease. The panD gene that enables Salmonella to synthesize β-alanine could serve as a potential target for new treatments or vaccines. By targeting this specific aspect of Salmonella's survival strategy, researchers may be able to develop more effective methods to prevent and treat these dangerous infections.

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Conflict of interest statement

SM, BY, YS, XW, HG, RL, TY, CK, JC, LJ No competing interests declared

Figures

Figure 1.
Figure 1.. 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 hr vs. 2 hr) 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).
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. The levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in RAW264.7 cells after infection with Salmonella WT for 8 hr, 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 a two-sided Student’s t-test. ns, not Significant.
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. 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 wild-type (WT) for 8 hr, 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.
Figure 1—figure supplement 3.
Figure 1—figure supplement 3.. Growth curves of Salmonella wild-type (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.
Figure 1—figure supplement 4.
Figure 1—figure supplement 4.. Growth curves of Salmonella wild-type (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.
Figure 1—figure supplement 5.
Figure 1—figure supplement 5.. Fold intracellular replication (20 hr vs. 2 hr) 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.
Figure 1—figure supplement 6.
Figure 1—figure supplement 6.. 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 the Mann-Whitney U test. ns, not Significant.
Figure 1—figure supplement 7.
Figure 1—figure supplement 7.. Growth curves of Salmonella wild-type (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.
Figure 1—figure supplement 8.
Figure 1—figure supplement 8.. Growth curves of Salmonella wild-type (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.
Figure 2.
Figure 2.. 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) Quantitative real-time PCR (qRT‒PCR) analysis of the expression of the Salmonella panD gene in RAW264.7 cells (8 hr post-infection) 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 hr vs. 2 hr) of Salmonella Typhimurium 14,028 s 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 hr post-infection. The number of intracellular bacteria per infected cell was estimated in random fields, n=80 cells per group from three independent experiments. (G) Representative immunofluorescence images of Salmonella WT, ΔpanD, and cpanD in RAW264.7 cells at 20 hr post-infection (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 a two-sided Student’s t-test (B–D) or one-way ANOVA (E, F, H). ns, not Significant.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Growth curves of Salmonella wild-type (WT), panD mutant (ΔpanD), and the complemented strain (cpanD) in LB medium.
Data are presented as mean ± SD, n=4 independent experiments.
Figure 2—figure supplement 2.
Figure 2—figure supplement 2.. Growth curves of Salmonella wild-type (WT), panD mutant (ΔpanD) and the complemented strain (cpanD) in RPMI-1640 medium (B).
Data are presented as mean ± SD, n=4 independent experiments.
Figure 2—figure supplement 3.
Figure 2—figure supplement 3.. Fold intracellular replication (20 hr vs. 2 hr) 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 a two-sided Student’s t-test. ns, not Significant.
Figure 3.
Figure 3.. 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 wild-type (WT), ΔpanD, or cpanD. n=10 randomly assigned mice per group. (D) Liver and spleen bacterial burdens and body weights of mice infected with Salmonella WT, ΔpanD, or cpanD on day 3 post-infection. n=7 randomly assigned mice per group. (E) Representative immunofluorescence images and intracellular bacterial counts of Salmonella WT, ΔpanD, and cpanD in mouse liver at 5 d 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 three 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 post-infection. 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).
Figure 4.
Figure 4.. 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 wild-type (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 Gene Ontology (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) Quantitative real-time PCR (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.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Expression of SPI-2 is shown in the Z-score transformed heatmap, with orange representing higher and blue representing lower abundance.
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Expression of SPI-1 is shown in the Z-score transformed heatmap, with orange representing higher and blue representing lower abundance.
Figure 4—figure supplement 3.
Figure 4—figure supplement 3.. 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.
Figure 5.
Figure 5.. 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 wild-type (WT), ΔfadAB, ΔmetR, ΔhisABCDFGHL, ΔkdpABC, ΔmglABC, ΔpotFGHI, or ΔleuO on day 3 post-infection. 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 post-infection. n=5 mice per group. (E) The zinc levels in the livers of mice infected with either Salmonella WT or ΔpanD for 3 d, 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.
Figure 6.
Figure 6.. 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 wild-type (WT) or ΔpanD for 8 hr. (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 a 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.
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Update of

  • doi: 10.1101/2024.10.07.616983
  • doi: 10.7554/eLife.103714.1
  • doi: 10.7554/eLife.103714.2
  • doi: 10.7554/eLife.103714.3

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