The putative AraC-type transcriptional regulator STM1082 facilitates Salmonella pathogenicity through modulation of intestinal invasion and intracellular replication

Shuai Ma; Wanwu Li; Xinyue Wang; Yuyang Sun; Houliang Guo; Jiaqi Ma; Lingyan Jiang

doi:10.1080/21505594.2025.2554297

. 2025 Sep 3;16(1):2554297. doi: 10.1080/21505594.2025.2554297

The putative AraC-type transcriptional regulator STM1082 facilitates Salmonella pathogenicity through modulation of intestinal invasion and intracellular replication

Shuai Ma ^a,^*, Wanwu Li ^b,^*, Xinyue Wang ^a, Yuyang Sun ^a, Houliang Guo ^a, Jiaqi Ma ^b, Lingyan Jiang ^a,^✉

PMCID: PMC12413039 PMID: 40899617

ABSTRACT

Salmonella is a food-borne zoonotic pathogen that causes human gastroenteritis and potentially fatal systemic infections. The invasion of intestinal epithelial cells and the survival and replication within host macrophages are essential for the intestinal and systemic infections caused by Salmonella, respectively. To precisely regulate the expression of virulence-related genes, Salmonella relies on a series of regulatory proteins. In this study, we identified STM1082, a putative AraC-type transcriptional regulator, as a new virulence activator in Salmonella. The expression of the STM1082 gene is up-regulated when Salmonella invades HeLa epithelial cells and replicates within RAW264.7 macrophages. Mutation of STM1082 impaired the ability of Salmonella to invade HeLa cells and replicate in RAW264.7 cells, and reduced its colonization in the mouse intestine, liver, and spleen. These results suggest that STM1082 facilitates Salmonella pathogenicity by promoting both its intestinal and systemic infections. Moreover, under intestinal-mimicking conditions, STM1082 increased the expression of genes related to 1,2-propanediol and ethanolamine metabolism, which are linked to Salmonella‘s intestinal survival. Under macrophage-mimicking conditions, it upregulated genes involved in purine biosynthesis, associated with Salmonella‘s systemic virulence. Together, these findings highlight the significant role of STM1082 in modulating the pathogenic mechanisms of Salmonella.

KEYWORDS: Salmonella, AraC-type transcriptional regulator, epithelial invasion, intracellular replication, pathogenicity

Introduction

Salmonella is a predominant food-borne pathogen capable of triggering a diverse spectrum of diseases in both humans and a variety of animals, ranging from self-limiting gastroenteritis to life-threatening systemic infections [1–3]. The severity of an infection in humans varies depending on the serovar of the infecting Salmonella and the immunological status of the host [4]. Non-typhoidal serovars, such as Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Enteritidis (S. Enteritidis), typically induce mild gastroenteritis in immunocompetent hosts [5]. In contrast, typhoidal serovars (S. Typhi and S. Paratyphi) cause fatal typhoid fever with elevated mortality, accounting for approximately 11–20 million annual global infections and 128,000–161,000 deaths [6]. Transmission of Salmonella usually occurs via contaminated food or water [7]. Once inside the host, Salmonella invades the intestinal epithelial cells, breaches the intestinal barrier, and is subsequently engulfed by phagocytes (such as macrophages and neutrophils) [8]. It then spreads to systemic organs like the liver and spleen via the bloodstream and lymphatic system [9]. In liver and spleen of a host, Salmonella primarily resides and replicates within the macrophages. The replication of Salmonella in macrophages within the liver and spleen triggers the accumulation of large numbers of bacteria, which can be released back into the bloodstream and eventually result in the widespread dissemination of the pathogen to secondary infection sites [10,11].

The two hallmarks of Salmonella pathogenesis are the invasion of epithelial cells in the intestinal mucosa and the survival and replication within host macrophages [12–15]. The intestinal invasion and intracellular replication of Salmonella are driven by the bacteria and rely on the expression of specific bacterial virulence factors. Salmonella pathogenicity islands (SPI)-1 and SPI-2, which encode two functionally distinct type III secretion systems (T3SS) that transfer effector proteins into host cells, are required for Salmonella‘s invasion of intestinal epithelial cells and replication within macrophages, respectively [12,16–18]. Apart from the effectors encoded by SPI-1 and SPI-2, numerous other virulence factors are also indispensable for Salmonella to cause diseases. These include proteins involved in the uptake, metabolism, and transport of nutrients [19–22], proteins contributing to oxidative stress resistance [23–25], and regulatory factors that participate in the regulation of bacterial virulence factors and/or host immunity [26–29].

Bacterial regulatory proteins encompass a highly diverse and extensive cohort of DNA-binding proteins that govern the initiation of transcription of target genes [30,31]. These regulatory factors confer bacterial pathogens with the capacity to adapt to a wide array of environmental conditions during their pathogenic process. The genome of Salmonella encodes a multitude of regulatory proteins. In addition to those with well-defined regulatory functions, there are over 50 putative regulatory proteins [32]. For the majority of these putative regulatory proteins, their physiological functions and regulatory roles during Salmonella infection process remain unclear. In recent years, emerging evidence has demonstrated that several putative regulatory proteins are implicated in the pathogenicity of Salmonella. For instance, the putative regulator protein YhjC and STM2345 (PagR) have been shown to promote the systemic infection of Salmonella. YhjC exerts its effect by modulating the expression of the virulence factor SpvD, while STM2345 acts by upregulating the expression of SPI-2 genes [26,33]. The putative regulator protein STM4320 (VarN) also facilitates the systemic infection of Salmonella by upregulating the expression of SPI-2 genes [32]. These findings underscore the underlying significance of putative regulatory proteins in the pathogenesis of Salmonella.

The STM1082 gene in Salmonella encodes a putative AraC-type transcriptional regulatory protein, which consists of 259 amino acids and harbors a predicted DNA-binding helix-turn-helix motif. NCBI BLAST analysis reveals its presence in major human-pathogenic serovars, including S. Typhimurium, S. Typhi, S. Paratyphi, and S. Enteritidis, with >95% amino acid sequence identity across these strains, indicating high conservation. Analysis of previously published transcriptome data reveals that the expression of the STM1082 gene is up-regulated within macrophages and under high-salt stimulation [34], a condition that simulates the intestinal environment [35–38]. The upregulation of the STM1082 gene under these pathogenesis-relevant conditions implies that it may play a role in modulating the pathogenicity of Salmonella. In this study, we employed in vitro and in vivo infection assays, immunofluorescence, RNA sequencing (RNA-seq), quantitative real-time PCR (qRT-PCR), and other molecular techniques to investigate the role of STM1082 in Salmonella virulence. Our findings demonstrated that STM1082 is crucial for Salmonella‘s invasion of epithelial cells, its replication within macrophages, and its pathogenicity in mice. Further analysis has revealed that under conditions that mimic the intestinal environment, STM1082 triggers the upregulation of genes involved in the metabolism of 1,2-propanediol and ethanolamine. These metabolites could act as energy sources to promote Salmonella growth within the intestinal environment [39–42]. Additionally, under conditions that simulate the intracellular environment of macrophages, STM1082 increases the expression of genes related to the biosynthesis of purine, a key component of nucleic acids, which may support the rapid replication of Salmonella inside macrophages [43].

Materials and methods

Ethics statement

All animal procedures were approved by the Institutional Animal Care Committee of Nankai University (Tianjin, China; approval ID: 2021-SYDWLL-000029) and adhered to the ARRIVE guidelines. Six-week-old female BALB/c mice, obtained from Beijing Vital River Laboratory Animal Technology in China, were utilized as the Salmonella infection model. The mice were housed in pathogen-free conditions within temperature-controlled facilities (maintained at 24 ± 2°C) under a 12-h light/dark cycle (with 50 ± 5% humidity). Animals received standardized rodent chow and water ad libitum throughout the experimental period.

Cell culture

The human cervix adenocarcinoma HeLa cell line (ATCC CCL-2) and the mouse macrophage-like RAW264.7 cell line (ATCC TIB-71) were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). Cultivation was performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco), under standardized conditions (37°C, 5% CO₂). For infection assays, 1 × 10⁵ cells/well were seeded in 24-well plates 24 h prior to experimental manipulation.

Bacterial strains, plasmids, and growth conditions

The S. Typhimurium strain ATCC 14028s (STM-WT) served as the genetic background for all engineered derivatives. The mutant strains were generated through λ red-recombinase-mediated homologous recombination using pSIM17 [44]. To construct the complemented strain of ΔSTM1082, the STM1082 ORF with its native promoter was PCR-amplified and cloned into the EcoRI-BamHI sites of the low-copy plasmid pBR322. The promoterless luxCDABE reporter plasmid pMS402 [45] was recombined with the STM1082 promoter region to construct reporter fusion STM1082-lux. All bacterial strains and plasmids used in this study are listed in Supplementary Table S1, and primer sequences for genetic manipulations are provided in Supplementary Table S2. All genetic constructs were confirmed by colony PCR and gene sequencing.

Bacterial strains were routinely cultivated in Luria–Bertani (LB) medium (containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl), N-minimal medium (comprising 10 μM MgCl₂, 110 μM KH₂PO₄, 7.5 mM (NH₄)₂SO₄, 0.5 mM K₂SO₄, 5 mM KCl, 38 mM glycerol, and 0.1% [w/v] casamino acids) or M9 minimal medium (47.9 mm Na2HPO4, 17.2 mm KH2PO4, 8.55 mm NaCl, 18.7 mm NH4Cl). Cultivation was performed at 37°C with shaking at 180 rpm for liquid cultures, while solid LB agar (1.5% [w/v]) was used as a static culture medium. Antibiotic selection (25 μg/mL chloramphenicol, 50 μg/mL kanamycin, 100 μg/mL ampicillin, 10–100 μg/mL gentamicin) was applied when required for strain selection.

Growth curve

Bacterial strains were initially cultured in LB medium and grown overnight at 37°C with shaking at 180 rpm. For aerobic kinetic growth analysis, sub-cultivation was performed at a 1:100 dilution ratio in both LB and RPMI-1640 medium. For anaerobic kinetic growth analysis, sub-cultivation was performed at a 1:100 dilution ratio in M9 minimal medium with 5 mM 1,2-propanediol or 5 mM ethanolamine as the sole carbon source, and with 40 mM sodium tetrathionate as respiratory electron acceptor [46,47]. Then, 200 μl of the bacterial inoculum was transferred to a 96-well white assay plate (Corning), followed by kinetic monitoring of the optical density at 600 nm (OD₆₀₀) using a Spark® multimode microplate reader (Tecan Group Ltd.). The growth kinetics were recorded at 30-min intervals over a 12-h experimental duration under quality-controlled parameters (37°C ± 0.5°C, constant shaking).

Salmonella invasion of HeLa epithelial cells

Bacterial cultures were established through 16-h incubation (37°C, 180 rpm) in LB medium until reaching stationary phase. For invasion assays, cultures were diluted 1:100 into fresh LB medium and subcultured at 200 rpm until mid-log phase (OD₆₀₀ ~ 0.6). The bacterial culture was then diluted to a concentration of 2 × 10⁶ colony-forming units (CFUs)/mL in RPMI-1640 medium containing 10% heat-inactivated FBS. Next, the bacterial culture was distributed on LB agar plates for CFU enumeration (input). Meanwhile, the bacterial culture was added to HeLa cell monolayers at a volume of 0.5 mL/well, with a multiplicity of infection (MOI) of 10. The infected cells were centrifuged at 1,000 × g for 5 min, followed by incubation in 5% CO₂ (37°C, 1 h). After incubation, the infected cells were washed three times with 1 × phosphate-buffered saline (PBS). Fresh RPMI-1640 medium supplemented with 100 μg/mL gentamicin was then added and incubated for 1 h to eliminate extracellular bacteria. Subsequently, the infected cells were lysed using 1% Triton X-100. The intracellular bacteria were then distributed on LB agar plates to enumerate the bacterial CFUs (output). The invasion of bacterial strains was calculated as the output/input CFU ratio, normalized to the number of HeLa cells.

Salmonella infection of macrophages

Bacterial strains were initially cultured in LB medium (37°C, 180 rpm) to stationary phase. Following opsonization in RPMI-1640 containing 10% heat-inactivated FBS for 15 min at 37°C, bacterial cultures were then diluted to a concentration of 2 × 10⁶ and added to RAW264.7 monolayers at 0.5 mL/well (MOI 10). The infected cells were centrifuged at 1,000 × g for 5 min and then incubated in 5% CO₂ (37°C, 30 min). After incubation, the infected cells were washed three times with 1 × PBS. Fresh RPMI-1640 medium supplemented with 100 μg/mL gentamicin was then added and incubated for 1 h to eliminate extracellular bacteria. Subsequently, the medium was replaced with RPMI-1640 medium supplemented with 10 μg/mL gentamicin for the remainder of the experiment. At the specified time points post-infection, intracellular bacteria were enumerated via 1% Triton X-100 lysis and LB agar plating. Intracellular replication of the bacterial strains was calculated as the ratio of CFUs recovered at 20 h post-infection (hpi) to 2 hpi, normalized to the number of RAW264.7 cells.

Bioluminescent reporter assays

The lux bioluminescent reporter system was implemented to quantify the expression of STM1082 in intestinal environment and within macrophages. Salmonella WT strain harboring the STM1082-lux transcriptional fusion were initially cultured in LB medium (37°C, 16 h, with aeration at 180 rpm). Subsequently, the overnight-grown bacteria were diluted 1:100 into either LB medium or N-minimal medium. Moreover, the bacteria infected HeLa cells or RAW264.7 cells following the method described previously. At indicated time points, 200 μl of the bacterial culture or cell lysate was transferred into a 96-well black assay plate (Corning). The measurement of luminescence was then carried out using the Spark multimode microplate reader (Tecan). To normalize the expression level of STM1082, the extracellular and intracellular bacterial CFUs were enumerated. Luminescence values were normalized to the bacterial CFUs.

Immunofluorescence staining

RAW264.7 macrophages were cultured on glass coverslips (1 × 10⁵ cells/well) and infected with Salmonella WT, ΔSTM1082 mutant, or the complemented strain cSTM1082 as described above. At 2 and 20 hpi, the infected cells were fixed with 4% paraformaldehyde for 15 min, followed by three washes with 1 × PBS. Subsequently, the cells were permeabilized with 1% Triton X-100 for 15 min, followed by blocking with 5% bovine serum albumin (BSA) in 1 × PBS for 30 min. After blocking, the infected cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-Salmonella antibody (diluted 1:100; Abcam) for 1 h in the dark. Then, the cells were incubated with 4,’6-diamidino-2-phenylindole (DAPI; Invitrogen) for 2 min. Confocal imaging was performed using Zeiss LSM800 system with ZEN 2.3 software for image analysis. Subsequently, Fiji-ImageJ software was utilized to estimate the number of intracellular bacteria in each infected cell within randomly selected fields.

RNA isolation

RNA (extracted from extracellular Salmonella) was isolated from the WT strain cultured in either N-minimal medium or in LB medium supplemented with 0.17 or 0.3 M NaCl. RNA (extracted from intracellular Salmonella) was isolated from bacteria within cells at 8 h post-infection of RAW264.7 cells or 1 h post-invasion of HeLa cells. Total RNA was purified using the EASYspinPlus Bacterial RNA Rapid Extraction Kit (Aidlab), following the manufacturer’s guidelines. The quantity and purity of the RNA were assessed with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies). The RNA samples were stored at −80°C until further analysis.

Quantitative real-time PCR (qRT-PCR)

Reverse transcription was performed using the StarScript III First-Strand Synthesis System (Genstar) with 1 μg of total RNA as the template. qRT-PCR was conducted in 20 μL reactions containing 2 × RealStar Power SYBR qPCR Mix (Genstar) on the QuantStudio 5 Real-Time PCR System (Applied Biosystems). Data were normalized to 16S rRNA expression, and relative gene expression was calculated by the 2^{− ΔΔCt} method.

RNA sequencing and analyses

Total RNA was extracted from STM WT and ΔSTM1082 cultures grown in either N-minimal medium for 8 h or high-salt LB medium containing 0.3 M NaCl for 2.5 h. Sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s instructions. High-throughput sequencing was performed on the Illumina HiSeq 2000 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.), and the raw data were deposited in the NCBI SRA database (PRJNA1247279). To identify differentially expressed genes (DEGs) between the STM1082 mutant and the WT, clean reads were mapped to the STM ATCC 14028s reference genome (CP001363/CP001362) using Bowtie 2. Transcript quantification was done using the fragments per kilobase of transcript per million mapped reads (FPKM) method. DEGs were identified using EdgeR in the R statistical framework. DEGs were considered significant with |log2(fold change)| ≥ 1 and false discovery rate (FDR)-adjusted p-values ≤0.05 (Benjamini–Hochberg correction). Functional annotation of DEGs involved Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.

Mouse infection

Oral infection. Salmonella strains (WT, ΔSTM1082 mutant, and complemented strain cSTM1082) were cultured in LB medium at 37°C with 180 rpm aeration until the stationary phase. Exponentially growing cultures (OD₆₀₀ ~ 0.6) were obtained by 1:100 sub-culturing in fresh LB medium with 200 rpm aeration. 1 × 10⁸ CFU of logarithmic-phase bacteria were orally administered to BALB/c mice (n ≥ 5/group). After 5 d of infection, the mice were euthanized after weight monitoring. Then, the liver, spleen, and cecum were collected, homogenized in ice-cold PBS, serially diluted, and plated on LB plates with appropriate antibiotics to determine CFUs/gram of tissue.

Intraperitoneal (i.p.) infection. Salmonella strains (WT, ΔSTM1082 mutant, and complemented strain cSTM1082) were cultured in LB medium at 37°C with 180 rpm aeration until the stationary phase. The stationary-phase bacteria were resuspended in 0.9% NaCl to a concentration of 5 × 10⁴ CFU/mL and injected intraperitoneally (i.p.) into BALB/c mice (0.1 mL per mouse). For survival analysis, infected mice were monitored daily for 10 d for body weight changes and mortality. To measure the bacterial load in the liver and spleen, infected mice were weighed 3 d post- infection (dpi) and then euthanized. The liver and spleen were collected, homogenized in ice-cold PBS, serially diluted, and plated on LB plates with appropriate antibiotics to quantify CFUs per gram of tissue. At 5 dpi, histopathological evaluation of the liver was conducted. Mice were euthanized, and the liver was excised, rinsed with 0.9% NaCl, and fixed in 10% neutral formalin for 48 h. The liver tissue was then embedded in paraffin, sliced into 5-μm-thick sections, and stained with H&E (hematoxylin and eosin). Stained sections were observed under a Leica DM2500 LED light microscope.

Statistical analysis

The data are presented as mean ± standard deviation (SD). All in vitro experiments were conducted in duplicate with at least three independent biological replicates (n ≥ 3). For the animal studies, two independent experiments were carried out, with at least two mice (n ≥ 2) in each injection group, and the pooled data were then subjected to statistical analysis. The statistical evaluations were performed using GraphPad Prism version 9.5.1 (GraphPad Software Inc., San Diego, CA). Depending on the test requirements (as specified in the figure legends), the appropriate statistical tests were chosen, including the two-sided Student’s t-test, one-way analysis of variance (ANOVA), two-way ANOVA, log-rank Mantel–Cox test for survival analysis, or the Mann-Whitney U test for non-parametric comparisons. Statistical significance was defined as p < 0.05.

Results

The expression of the STM1082 gene is up-regulated under conditions related to the intestinal environment and within macrophages

To determine if the expression of the STM1082 gene is up-regulated under conditions related to the intestinal environment and within macrophages, as indicated by published transcriptome data [34], we conducted quantitative real-time PCR (qRT‒PCR) and bioluminescent reporter assays. The qRT‒PCR results demonstrated that during the invasion of HeLa epithelial cells, the expression of STM1082 was increased by 1.7-fold compared to that in RPMI medium (Figure 1(a), p < 0.01). When cultured in a high-salt LB medium containing 0.3 M NaCl, which mimics the physiological conditions of the intestine [35–38], the expression of STM1082 was elevated by 1.8-fold relative to that in LB medium containing 0.17 M NaCl (Figure 1(a), p < 0.05). Bioluminescent reporter assays also confirmed the increased expression of STM1082 gene during the invasion of in HeLa epithelial cells and under high-salt stimulation (Figure 1(b), p < 0.01). These data reveal that STM1082 gene expression is up-regulated under conditions related to the intestinal environment.

In mouse RAW264.7 macrophages, the expression of STM1082 gene was up-regulated by 2.2-fold when compared with that in RPMI medium (Figure 1(c), p < 0.001). Additionally, in N-minimal medium, which mimics the conditions within macrophages [37,38,48,49], the expression of STM1082 was increased by 3.2-fold compared to that in LB medium (Figure 1(c), p < 0.01). Bioluminescent reporter assays also confirmed the increased expression of STM1082 gene within RAW264.7 macrophages and in N-minimal medium ((Figure 1(d), p < 0.0001). These data clearly demonstrate that the expression of the STM1082 gene is up-regulated within macrophages.

Collectively, these findings suggest that STM1082 could be involved in regulating intestinal and systemic infections caused by Salmonella.

STM1082 is crucial for Salmonella invasion into epithelial cells and replication within macrophages

To further evaluate the role of STM1082 in intestinal and systemic infections caused by Salmonella, we generated an STM1082 mutant strain, ΔSTM1082. Given that the invasion of epithelial cells and the replication within macrophages are pivotal events in Salmonella-mediated intestinal and systemic infections, respectively, we then compared the invasion ability of ΔSTM1082 and the wild-type (WT) strain into HeLa epithelial cells, along with their replication ability in RAW264.7 macrophages.

Gentamicin protection assays indicated that, at 1 h post-infection, the invasion of ΔSTM1082 into HeLa cells was reduced by 2.5-fold compared to the WT strain (Figure 2(a), p = 0.0001). Regarding replication, at 20 h post-infection of RAW264.7 cells, the replication of ΔSTM1082 was decreased by 2.4-fold compared with the WT strain ((Figure 2(b), p = 0.0017). When the ΔSTM1082 mutant was complemented with the STM1082 gene, both the invasion ability of the mutant into HeLa cells and its replication ability within RAW264.7 macrophages were restored. Furthermore, the growth rates of the ΔSTM1082 strain in both LB medium and RPMI medium were similar to those of the WT strain (Figure S1(a, b)). This finding implies that the reduced invasive capacity and intracellular replication ability of the mutant are not attributable to a growth defect. Immunofluorescence analysis revealed that during the early phase of infection (2 h), the number of ΔSTM1082 within each infected RAW264.7 cell was comparable to that of the WT strain. Nevertheless, at 20 h post-infection, the number of ΔSTM1082 in each infected RAW264.7 cell was markedly lower (p = 0.0002) compared to that of the WT strain (Figure 2(c, d)).

Figure 2. — STM1082 is crucial for *Salmonella* invasion into epithelial cells and replication within macrophages. (a) Invasion of *Salmonella* WT, *STM1082* mutant (Δ*STM1082*) and the complemented strain (cSTM1082) in HeLa cells. (b) Replication of *Salmonella* WT, Δ*STM1082*, and cSTM1082 in RAW264.7 cells. (c) Number of intracellular *Salmonella* WT, Δ*STM1082*, and cSTM1082 in each RAW264.7 cell at 2 and and 20 h post-infection. The number of intracellular bacteria per infected cell was counted in random fields, n = 80 cells per group obtained from three independent experiments. (d) Representative immunofluorescence images of *Salmonella* WT, Δ*STM1082*, and cSTM1082 in RAW264.7 cells at 20 h post-infection (green, *Salmonella*; blue, nuclei; scale bars, 50 µm). The representative images come from three independent experiments. Data are shown as mean ± SD, n = 3 (a, b) independent experiments. Statistical significance was evaluated by one-way ANOVA (a-c).

Collectively, these findings indicate that the STM1082 gene plays a pivotal role in the invasion of Salmonella into epithelial cells and its replication within macrophages.

STM1082 is essential for the full virulence of Salmonella in mice

We next investigated the role of STM1082 in the in vivo virulence of Salmonella. BALB/c mice were subjected to infection either through oral administration (with a dose of 10⁸ CFUs) or via i.p. injection (with a dose of 5000 CFUs) using the WT strain, the ΔSTM1082 mutant, and the complemented strain cSTM1082. Oral infection mimics the natural route of Salmonella infection, enabling a direct assessment of the bacteria’s ability to colonize the intestines [50]. In contrast, i.p. injection provides a more direct introduction of the bacteria into the systemic circulation, bypassing the initial stages of gastrointestinal infection. This approach facilitates the assessment of Salmonella‘s capacity to trigger systemic infection [51,52].

In orally infected mice, we evaluated the bacterial load in the cecum and systemic tissues (liver and spleen), as well as the body weight of the infected mice, at 5 d post-infection (Figure 3(a)). We observed that the bacterial load of ΔSTM1082 in the cecum, liver, and spleen was notably lower (p < 0.0001) compared to that of the WT strain. Moreover, the body weight of mice infected with ΔSTM1082 was notably higher (p < 0.0001) than that of mice infected with the WT strain. When ΔSTM1082 was complemented with the STM1082 gene, the bacterial load and body weight of the infected mice were significantly restored to a level comparable to that of the WT strain (Figure 3(b)). These results suggest that STM1082 contributes to the intestinal infection of Salmonella in mice.

Figure 3. — STM1082 facilitates the intestinal infection of *salmonella* in mice. (a) a schematic illustration is presented to depict the mouse oral infection assays. The picture materials employed in this illustration were sourced from bioicons (https://bioicons.com/) or SciDraw (https://scidraw.Io/). (b) The bacterial load in cecum, liver and spleen bacterial burdens, and body weight of mice infected with *salmonella* WT, Δ*STM1082*, and cSTM1082, at day 5 post-infection. A total of 24 mice were randomly divided into 4 groups, with 6 mice in each group. Mice orally administered with an equivalent dosage of 0.9% NaCl were used as a negative control group (not shown in the figure). Data are shownas mean ± SD (b). Statistical significance was evaluated by using one-way ANOVA (b).

In mice infected via i.p. injection, we assessed the bacterial load in the liver and spleen, survival rate, body weight, and liver histopathological changes (Figure 4(a)). We found that during the 10-d surveillance period, mice infected with the WT strain had higher mortality and experienced more significant weight loss than those infected with the ΔSTM1082 strain (Figure 4(b, c)). All mice infected with the WT strain succumbed within 5 d post-infection. In contrast, the mortality rate of the ΔSTM1082-infected mice was only 30% during the 10-d surveillance period (Figure 4b). Consistent with these findings, at 3 d post-infection, the bacterial load in the liver and spleen of the ΔSTM1082-infected mice was significantly lower (p < 0.01), and their body weight was significantly higher (p < 0.0001) compared to the WT-infected mice (Figure 4(d)). When the ΔSTM1082 strain was complemented with the STM1082 gene, it significantly reduced the survival rate and body weight of the infected mice while markedly increasing the bacterial burden in their liver and spleen (Figure 4(b–d)). Furthermore, using hematoxylin and eosin (H&E) staining, we observed that there was an enhanced aggregation of inflammatory cells and pyknosis in the livers of WT-infected mice at 5 d post-infection. Conversely, these histopathological alterations were markedly diminished in the livers of mice infected with the ΔSTM1082 strain (Figure 4(e)). These results suggest that STM1082 facilitates the systemic infection of Salmonella in mice.

Figure 4. — STM1082 facilitates the systemic infection of *salmonella* in mice. (a) a schematic illustration is presented to depict the mouse intraperitoneal (i.P.) infection. The picture materials employed in this illustration were sourced from bioicons (https://bioicons.com/) or SciDraw (https://scidraw.Io/). (b, c) survival rate (b) and body weight changes (c) of mice infected i.P. With *salmonella* WT, Δ*STM1082*, and cSTM1082. A total of 40 mice were randomlydivided into 4 groups, with 10 mice in each group. Mice i.P. injected with equivalent dosage of 0.9% NaCl were used as a negative control group (not shown in the figure). (d) The bacterial load in liver and spleen bacterial burdens, and body weight of mice infected with *salmonella* WT, Δ*STM1082*, and cSTM1082, at day 3 post-infection. A total of 32 mice were randomly divided into 4 groups, with 8 mice in each group. Mice i.P. injected with equivalent dosage of 0.9% NaCl were used as a negative control group (not shown in the figure). (e) Representative H&E-stained liver sections from mice that left uninfected or infected with *salmonella* WT, Δ*STM1082*, and cSTM1082, at day 5 post-infection. Severe inflammatory cell infiltration in the mouse liver is indicated by the arrows. Images are typical of three independent experiments. Data are shown as mean ± SD (b-d). Statistical significance was evaluated by log-rank mantel–cox test (b), two-sided Student’s t-test (c), or one-way ANOVA (d).

Collectively, these results demonstrate that STM1082 is essential for the full virulence of Salmonella in mice, facilitating both the intestinal and systemic infections caused by Salmonella.

STM1082 up-regulates the expression of genes involved in 1, 2-propanediol and ethanolamine metabolism under intestinal-mimicking conditions

To investigate the potential mechanism by which STM1082 facilitates the intestinal infection of Salmonella, using RNA-seq, we determined the differences in gene transcripts between the WT and ΔSTM1082 strains cultured in high-salt LB medium containing 0.3 M NaCl, which simulates the intestinal environment [35–38]. Compared with the WT strain, 71 differentially expressed genes (DEGs) were identified in the ΔSTM1082 mutant, including 52 upregulated and 19 downregulated genes (fold change ≥ 2.0 and p < 0.05; Table 1, Figure 5(a)). Of the 52 upregulated DEGs (repressed by STM1082), 69% (36/52) correspond to putative proteins with incompletely defined functions. Besides, the upregulated DEGs also include SPI-4 gene siiA and the aromatic amino acid transporter yddG, suggesting that STM1082 represses these processes. Among the 19 downregulated DEGs (activated by STM1082), 47% (9/19) are enriched in pathways involved in 1,2-propanediol and ethanolamine metabolism, including genes from the pdu (pduD/E/K/U/J) and eut (eutN/P/Q/T) operons (Figure 5(b)), indicating that STM1082 activates these metabolic pathways. Strikingly, no significant differential expression was observed for pocR or eutR, the transcriptional regulators of the pdu and eut operons, respectively, suggesting that STM1082 may act either downstream of PocR/EutR or in a parallel regulatory layer to modulate pdu/eut gene expression. Notably, SPI-1 genes, which encode the key virulence locus required for Salmonella intestinal epithelial invasion [18,53], remained unaffected by the ΔSTM1082 mutation (Supplementary Table S3). This observation underscores STM1082’s distinct regulatory role in metabolic pathways rather than SPI-1-dependent invasion mechanisms.

Table 1.

Degs between the WT and ΔSTM1082 strains cultured in high-salt LB medium containing 0.3 M NaCl.

GeneName	log₂ fold change	p value	Regulation	Function
eutT	−2.07	< 0.0001	Down	putative ethanolamine utilization cobalamin adenosyltransferase
eutN	−2.33	0.0002	Down	ethanolamine utilization protein EutN
eutP	−1.00	0.0009	Down	putative ethanolamine utilization protein
yccX	−1.13	0.0009	Down	acylphosphatase
srfJ	−1.17	0.0026	Down	lysosomal glucosyl ceramidase-like type III secretion effector SrfJ
pduU	−1.02	0.0029	Down	propanediol utilization protein
pduD	−1.13	0.0060	Down	propanediol utilization dehydratase, medium subunit
pduE	−1.46	0.0092	Down	propanediol utilization dehydratase, small subunit
pduK	−1.99	0.0156	Down	propanediol utilization protein
araH	−1.23	0.0213	Down	putative intracellular protease/amidase
iolI1	−1.28	0.0221	Down	putative 4-hydroxyphenylpyruvate dioxygenase
iolI2	−2.81	0.0231	Down	putative 4-hydroxyphenylpyruvate dioxygenase
traR	−2.50	0.0235	Down	conjugal transfer protein TraR
allD	−2.38	0.0391	Down	ureidoglycolate dehydrogenase
yigG	−4.68	0.0435	Down	membrane protein
yiaN	−1.18	0.0443	Down	2%2C3-diketo-L-gulonate transporter large permease YiaN
eutQ	−0.98	0.0444	Down	putative ethanolamine utilization protein
pduJ	−1.50	0.0461	Down	propanediol utilization protein PduJ
STM3548	−1.35	0.0467	Down	glutamine amidotransferase
rfbI	2.30	0.0001	Up	GtrA family protein
ymcF	1.99	0.0001	Up	hypothetical protein
ffs	1.31	0.0001	Up	signal recognition particle sRNA small type
STM2242	1.20	0.0002	Up	tail protein
STM0065	1.73	0.0003	Up	hypothetical protein
STM2374	2.78	0.0003	Up	transcriptional regulator
STM3911	1.26	0.0004	Up	nicotinamide riboside transporter PnuC
STM2229	2.51	0.0005	Up	tRNA-Pro
STM0294	2.15	0.0006	Up	immunity 26/phosphotriesterase HocA family protein
proV	1.02	0.0007	Up	glycine betaine/L-proline ABC transporter ATP-binding protein ProV
cspB	1.07	0.0007	Up	cold-shock protein
yedE	1.03	0.0013	Up	selenium metabolism membrane protein YedE/FdhT
sfsB	1.05	0.0016	Up	DNA-binding transcriptional regulator SfsB
cueP	1.22	0.0017	Up	copper-binding periplasmic metallochaperone CueP
STM2329	1.73	0.0022	Up	hypothetical protein
yjfL	1.51	0.0023	Up	DUF350 domain-containing protein
yibR	2.25	0.0024	Up	glycosyltransferase family 8 protein
STM0159	1.63	0.0025	Up	HNH endonuclease
STM2156A	2.88	0.0025	Up	YehR family lipoprotein
malY	1.14	0.0034	Up	transcriptional regulator
STM3605	3.35	0.0045	Up	lysozyme
STM1585	1.03	0.0048	Up	YgdI/YgdR family lipoprotein
fic	1.25	0.0054	Up	cell filamentation protein Fic
STM2376	2.13	0.0059	Up	YbjP/YqhG family protein
rbsK	2.27	0.0059	Up	ribokinase
sirC	2.03	0.0059	Up	invasion transcriptional regulator HilC
STM2377	1.25	0.0066	Up	CPBP family intramembrane metalloprotease
pefI	1.19	0.0076	Up	transcriptional regulator PefI
ytfG	1.37	0.0080	Up	SDR family oxidoreductase
STM2508	1.31	0.0080	Up	DUF1493 family protein
STM0293	1.20	0.0081	Up	hypothetical protein
siiA	5.19	0.0082	Up	SPI-4 type I secretion system auxiliary protein SiiA
STM4011	1.10	0.0095	Up	STM4011 family radical SAM protein
dppF	1.09	0.0097	Up	dipeptide transport protein
glf	2.61	0.0098	Up	UDP-galactopyranose mutase
rprA	1.77	0.0121	Up	antisense sRNA RprA
csgC	1.09	0.0122	Up	putative curli production protein
yoeI	1.40	0.0131	Up	membrane protein YoeI
yddG	1.21	0.0137	Up	aromatic amino acid efflux DMT transporter YddG
STM0347	3.12	0.0143	Up	helix-turn-helix transcriptional regulator
STM4039	1.10	0.0147	Up	YiiG family protein
rseD	1.56	0.0169	Up	rpoE leader peptide RseD
ftsI	1.08	0.0176	Up	division specific transpeptidase
STM4449	1.27	0.0188	Up	type II toxin-antitoxin system RelB/DinJ family antitoxin
yqjF	1.71	0.0201	Up	DoxX family protein
STM2274	1.33	0.0210	Up	MFS transporter
asnW	1.26	0.0240	Up	tRNA-Asn
STM4390	2.39	0.0255	Up	hypothetical protein
rarD	1.34	0.0289	Up	EamA family transporter RarD
rfbX	1.22	0.0324	Up	MATE family efflux transporter
citD	4.61	0.0404	Up	citrate lyase acyl carrier protein
ybiV(2)	1.03	0.0407	Up	HAD family hydrolase

Open in a new tab

Figure 5. — STM1082 upregulates the expression of genes involved in 1,2-propanediol and ethanolamine metabolism under intestinal-mimicking conditions. (a) Volcano plot of the differentially expressed genes (DEGs) in *Salmonella* WT versus Δ*STM1082* cultured in high-salt LB medium (0.3 M NaCl). The upper right section (red dots) indicates the upregulated genes, and the upper left section (blue dots) indicates downregulated genes. (b) Expression of the significantly downregulated pathways (activated by STM1082) is shown with the log₂ Fold change. Data are derived from RNA sequencing of a pooled sample generated from three independent biological experiments (a,b). (c) qRT-PCR analyzes the mRNA levels of 6 selected downregulated genes in *Salmonella* WT, Δ*STM1082*, and cSTM1082. (d) qRT-PCR analyzes the mRNA levels of *Salmonella pduD/E/K* (left) and *eutN/P/Q* (right) genes in WT and Δ*STM1082* under inducing conditions (LB medium supplemented with 5 mM 1,2-propanediol or 5 mM ethanolamine) or non-inducing conditions (without 1,2-propanediol or ethanolamine). (e) Growth curves of *Salmonella* WT and the *STM1082* mutant (Δ*STM1082*) in M9 minimal medium supplemented with 5 mM 1,2-propanediol or 5 mM ethanolamine as the sole carbon source. Data are shown as mean ± SD, n = 3 independent experiments. Statistical significance was evaluated by two-way ANOVA.

To confirm the RNA-seq findings, we selected 6 down-regulated genes, including pduD/E/K (related to 1, 2-propanediol metabolism) and eutN/P/Q (related to ethanolamine metabolism), for qRT-PCR analysis. The results demonstrated that, in comparison to the WT strain, the expression of these six genes was markedly reduced (p < 0.01) in the ΔSTM1082 mutant. When the ΔSTM1082 mutant was complemented with the STM1082 gene, the gene expression returned to the WT level (Figure 5(c)). These data confirmed the positive regulation of 1, 2-propanediol and ethanolamine metabolism by STM1082.

To further investigate STM1082’s role in activating these pathways, we analyzed pduD/E/K and eutN/P/Q expression in WT and ΔSTM1082 under inducing conditions (LB medium supplemented with 5 mM 1,2-propanediol or 5 mM ethanolamine). In inducing conditions, ΔSTM1082 exhibited severely impaired gene induction: pduD/E/K expression was reduced 4.4- to 9.9-fold (p < 0.0001), and eutN/P/Q expression was diminished 3.9- to 5.7-fold (p < 0.0001) (Figure 5(d)). Under non-induced LB conditions (no added inducers), pdu/eut gene expression in ΔSTM1082 showed only modest reductions (2.0- to 2.5-fold) (Figure 5(d)). These data indicate that STM1082 is critical for robust induction of both metabolic pathways specifically when their inducers are present.

Salmonella can utilize 1, 2-propanediol and ethanolamine as carbon sources to support its growth within the intestinal environment [39–42]. To assess STM1082’s functional role, we compared the growth of WT and ΔSTM1082 using either 1,2-propanediol or ethanolamine as the sole carbon source. The ΔSTM1082 mutant exhibited markedly impaired growth under both conditions (Figure 5(e)), confirming that STM1082 is critical for efficient utilization of these carbon sources.

Integrating RNA-seq, qRT-PCR, and growth assay data, we propose that STM1082 promotes the intestinal infection of Salmonella by enhancing the utilization of 1, 2-propanediol and ethanolamine. Upon Salmonella‘s entry into the intestinal environment, STM1082 is up-regulated, activating the genes involved in the metabolic pathways of 1, 2-propanediol and ethanolamine. This regulatory cascade is speculated to provide Salmonella with an adequate supply of carbon sources, thereby promoting its growth and infection within the intestinal tract.

STM1082 up-regulates the expression of genes involved in purine biosynthesis under macrophage-mimicking conditions

We next explored the underlying mechanism(s) by which STM1082 facilitates Salmonella intracellular replication and systemic infection. Using RNA-seq, we determined the differences in gene transcripts between the WT and ΔSTM1082 strains cultured in N-minimal medium, which resembles the conditions inside macrophages [37,38,48,49]. In comparison with the WT strain, 38 DEGs were identified in the ΔSTM1082 mutant, including 18 up-regulated genes and 20 down-regulated genes (fold change ≥ 2.0 and p value < 0.05; Table 2, Figure 6(a)). Among the upregulated DEGs (repressed by STM1082), 6 correspond to putative protein-coding genes, alongside genes associated with phage shock response (pspC/D) and polymyxin resistance (arnA/B/C). The downregulated DEGs (activated by STM1082) are enriched in pathways linked to purine biosynthesis (purE/C/M), nitrite utilization (napA/D/F, narH/J/K) and citrate utilization (citC/D/F/E) (Figure 6(b)), indicating STM1082 activate these processes under macrophage-mimicking conditions.

Table 2.

Degs between the WT and ΔSTM1082 strains cultured in N-minimal medium.

GeneName	log₂ fold change	p value	Regulation	Function
frwC	−1.07	< 0.0001	Down	helix-turn-helix domain-containing protein
cbiA	−1.29	< 0.0001	Down	cobyrinate a%2Cc-diamide synthase
napD	−1.25	< 0.0001	Down	periplasmic nitrate reductase
citC	−1.97	< 0.0001	Down	citrate lyase synthetase (citrate (pro-3S)-lyase ligase
napA	−1.05	< 0.0001	Down	nitrate reductase catalytic subunit NapA
citD	−1.79	< 0.0001	Down	citrate lyase acyl carrier protein (gamma chain)
napF	−1.04	< 0.0001	Down	ferredoxin-type protein NapF
narH	−1.12	< 0.0001	Down	nitrate reductase subunit beta
purC	−1.27	< 0.0001	Down	propanediol dehydratase large subunit PduC
csgE	−1.86	< 0.0001	Down	curli production assembly/transport component, 2nd curli operon
purM	−1.32	< 0.0001	Down	phosphoribosylaminoimidazole synthetase
csgC	−1.27	< 0.0001	Down	putative curli production protein
purE	−1.25	< 0.0001	Down	phosphoribosylaminoimidazole carboxylase = AIR carboxylase, catalytic subunit
nirD	−1.09	< 0.0001	Down	nitrite reductase small subunit
nirC	−1.03	< 0.0001	Down	FNT family nitrite transport protein
citF	−1.12	< 0.0001	Down	citrate lyase subunit alpha
csgB	−1.02	< 0.0001	Down	minor curlin subunit precursor
narK	−1.21	< 0.0001	Down	nitrate transporter NarK
narJ	−1.14	< 0.0001	Down	nitrate reductase molybdenum cofactor assembly chaperone
citE	−1.01	< 0.0001	Down	citrate (pro-3S)-lyase subunit beta
rcsA	1.17	< 0.0001	Up	positive transcriptional regulator of capsular/exo- polysaccharide synthesis (LuxR/UhpA family)
arnC	1.37	< 0.0001	Up	undecaprenyl-phosphate 4-deoxy-4-formamido-L-arabinose transferase
cigR	1.12	< 0.0001	Up	membrane protein
arnB	1.07	< 0.0001	Up	UDP-4-amino-4-deoxy-L-arabinose aminotransferase
arnA	1.18	< 0.0001	Up	bifunctional UDP-4-amino-4-deoxy-L-arabinose formyltransferase/UDP-glucuronic acid oxidase ArnA
carB	1.02	< 0.0001	Up	carbamoyl-phosphate synthase large subunit
aceE	1.12	< 0.0001	Up	pyruvate dehydrogenase, decarboxylase component
mgtA	1.37	< 0.0001	Up	magnesium-translocating P-type ATPase
wcaE	1.28	< 0.0001	Up	putative transferase
pspC	1.00	< 0.0001	Up	phage shock protein
STM3521	1.15	< 0.0001	Up	TROVE domain-containing protein
STM1634	1.07	< 0.0001	Up	putative ABC transporter permease
STM1635	1.08	< 0.0001	Up	amino acid ABC transporter ATP-binding protein
eptA	1.23	< 0.0001	Up	phosphoethanolamine transferase EptA
pspD	1.02	< 0.0001	Up	phage shock protein PspD
STM4066	1.08	< 0.0001	Up	aminoimidazole riboside kinase
yjbF	1.02	< 0.0001	Up	YjbF family lipoprotein
STM0557	1.04	0.0001	Up	membrane protein

Open in a new tab

Figure 6. — STM1082 upregulates the expression of genes involved in purine biosynthesis under macrophage-mimicking conditions. (a) Volcano plot of the differentially expressed genes (DEGs) in *Salmonella* WT versus Δ*STM1082* cultured in N-minimal medium. The upper right section (red dots) indicates the upregulated genes, and the upper left section (blue dots) indicates downregulated genes. (b) Expression of the significantly downregulated pathways (activated by STM1082) is shown in the log₂ Fold change transformed heatmap. (c) qRT-PCR analyze the mRNA levels of downregulated genes *purC/E/M* in *Salmonella* WT, Δ*STM1082*, and cSTM1082. (d) Replication of *Salmonella* WT and Δ*purE* in RAW264.7 cells. (e) The bacterial load in liver and spleen bacterial burdens, and body weight of mice infected with *Salmonella* WT and Δ*purE* at day 3 post-infection. n = 5 mice in each group. Data are shown as mean ± SD, n = 3 independent experiments (c, d). Statistical significance was evaluated by two-way ANOVA (c), or two-sided Student’s t-test (d, e).

We used qRT-PCR analysis to validate the RNA-seq data. The results demonstrated that, in comparison to the WT strain, the expression of purE, purC, and purM was markedly (p < 0.0001) reduced in the ΔSTM1082 mutant. When the ΔSTM1082 mutant was complemented with the STM1082 gene, the gene expression returned to the WT level (Figure 6(c)). These findings confirmed that STM1082 positively regulates purine biosynthesis.

Previous research has documented that mutation of purE gene, one of the genes participating in the purine biosynthetic pathway [43], results in a marked attenuation of Salmonella virulence in cattle following oral infection [54] and reduced colonization in the livers and spleens of mice following intravenous infection [54,55]. Based on the RNA-seq and qRT-PCR analysis, we hypothesize that STM1082 might facilitate the intracellular replication and systemic infection of Salmonella via the up-regulation of purE gene expression. To validate this hypothesis, we generated a purE mutant strain, ΔpurE. Gentamicin protection assays showed that the replication of ΔpurE in RAW264.7 macrophages was decreased by 3.2-fold, when compared with the WT strain (Figure 6(d), p < 0.0001). This finding suggests that purE plays a role in promoting Salmonella replication within macrophages. Mouse infection assays revealed that following i.p infection, Salmonella colonization in the mouse liver and spleen was markedly (p < 0.0001) reduced due to the mutation of purE (Figure 6(e)). Moreover, the body weight of ΔpurE-infected mice was markedly (p < 0.001) higher than that of the WT-infected mice (Figure 6(e)). These results imply that purE contributes to Salmonella systemic infection in mice. Collectively, these findings suggest that STM1082 may enhance Salmonella intracellular replication and systemic infection by activating the expression of purE and the associated purine biosynthetic pathway.

Discussion

The invasion of host intestinal epithelial cells and subsequent replication within host macrophages are pivotal events in Salmonella infection [12–15]. Salmonella employs transcription regulators to precisely govern its invasion and replication processes within the host [56,57]. To date, numerous transcription regulatory proteins have been identified. These proteins directly or indirectly modulate virulence genes (located on Salmonella pathogenicity islands [SPIs] and plasmids) [26–29], effectors [58,59], other transcription regulators [18], and even themselves in Salmonella [18], thereby playing a significant role in the pathogenesis of this bacterium. In this study, we demonstrated that the putative AraC-type transcription regulator STM1082 can enhance the ability of Salmonella to invade intestinal epithelial cells and replicate within macrophages. Specifically, we revealed that STM1082 might facilitate the intestinal infection of Salmonella by enhancing the utilization of 1, 2-propanediol and ethanolamine in the host intestine. Additionally, STM1082 might promote the intracellular replication and systemic infection of Salmonella by up-regulating the expression of genes involved in purine synthesis. In summary, our work has characterized a novel Salmonella transcription regulator that can influence both the intestinal invasion and intracellular replication of Salmonella.

STM1082 is a putative AraC-type transcription regulator. AraC-type transcription regulators are known to play crucial roles in diverse bacterial physiological processes, including carbon metabolism, stress response, and the establishment of pathogenesis [60]. In Salmonella, several AraC-type transcription regulators have been verified to participate in the regulation of virulence. For example, HilC, HilD, RstA, and InvF, all members of the AraC-type transcription regulator family, enhance Salmonella‘s pathogenicity by facilitating its invasion into the intestinal epithelial cells [56,61]. Additionally, RamA, another member of this class of transcription regulators, promotes Salmonella‘s intracellular replication and the subsequent systemic infection [57]. In the present study, we observed that the mutation of the STM1082 gene resulted in a notable decrease in Salmonella‘s ability to invade epithelial cells and colonize the mouse cecum. This finding indicates that STM1082 positively regulates the early-stage invasion of Salmonella into the host. Furthermore, the mutation of the STM1082 gene also led to a decrease in Salmonella‘s replication within host macrophages and its capacity to induce systemic infection in mice. This implies that STM1082 positively regulates the later stage of Salmonella‘s survival and replication within the host. Therefore, our results demonstrate that the putative AraC-type transcription regulator STM1082 is of exceptional significance in the pathogenicity of Salmonella.

We demonstrated that the mutation of the STM1082 gene down-regulates the expression of several genes involved in the metabolism of 1, 2-propanediol and ethanolamine in high-salt LB medium, which simulates the intestinal environment [35–38]. 1, 2-propanediol is a metabolite of L-rhamnose and L-fucose in the intestine [62]. During its catabolic process, it is initially converted into propionaldehyde [63]. Ethanolamine, present in the intestine, is derived from phosphatidylethanolamine, a major component of eukaryotic membranes [64–66]. In its catabolism, it is first transformed into acetaldehyde [67]. High concentrations of propionaldehyde and acetaldehyde can strongly inhibit cell viability [67,68]. Nevertheless, the growth of Salmonella is not inhibited by the propionaldehyde and acetaldehyde produced during the catabolism of 1, 2-propanediol and ethanolamine. Notably, Salmonella can utilize these two substances as carbon and energy sources [39–42]. This phenomenon can be attributed to the fact that the genes involved in the metabolism of 1, 2-propanediol and ethanolamine in Salmonella encode the Pdu and Eut microcompartments (MCPs), respectively [69]. The Pdu MCPs and Eut MCPs are proteinaceous shells that encapsulate a multitude of proteases [69,70]. These MCPs sequester propionaldehyde and acetaldehyde and ultimately convert them into non-cytotoxic alcohols and acids that can be readily utilized by Salmonella [71,72]. Consequently, the utilization of 1, 2-propanediol and ethanolamine by Salmonella can enhance its competitiveness and pathogenicity within the host intestine. Previous studies have demonstrated that the utilization of 1,2-propanediol and ethanolamine enhances the proliferation and colonization of Salmonella in the inflamed intestine [46,47]. Our research reveals that STM1082 positively regulates the expression of genes related to 1, 2-propanediol and ethanolamine metabolism. Given the crucial role of 1, 2- propanediol and ethanolamine utilization in Salmonella‘s intestinal infection, it is highly plausible that STM1082 promotes Salmonella‘s intestinal infection by enhancing the utilization of 1, 2-propanediol and ethanolamine.

Our cell invasion assays demonstrate that STM1082 promotes Salmonella‘s ability to invade epithelial cells. Interestingly, while SPI-1 genes are essential for epithelial cell invasion [16], our RNA-seq data revealed no significant differences in SPI-1 gene expression between WT and ΔSTM1082 strains under high-salt LB conditions. This finding suggests that STM1082 does not overtly regulate SPI-1 expression under these in vitro conditions. The disconnect between STM1082’s invasion-promoting function and its lack of effect on SPI-1 expression may arise from limitations of in vitro culture models, which often fail to fully recapitulate the complex host microenvironment encountered during infection. It is possible that STM1082 influences invasion through post-transcriptional modulation of SPI-1 effectors, regulatory effects under host-specific conditions (e.g. contact with epithelial cells), or interactions with other virulence factors not captured in our RNA-seq data. Future studies using infection-mimicking models (e.g. co-culture with epithelial cells) will be critical to elucidate the precise molecular basis of STM1082’s invasion-enhancing activity.

Notably, STM1082 enhances Salmonella invasion of epithelial cells, which in turn triggers intestinal inflammation [73]. During the inflammatory response, intestinal tetrathionate production enables Salmonella to utilize 1,2-propanediol and ethanolamine as alternative carbon sources, conferring a competitive edge in the gut [46,74]. Concurrently, STM1082 upregulates genes involved in 1,2-propanediol and ethanolamine metabolism, further augmenting bacterial exploitation of these carbon sources. Collectively, STM1082’s promotion of invasion and its enhancement of metabolic gene expression establishes a self-reinforcing cycle that amplifies Salmonella fitness in the inflamed intestine.

We also demonstrated that the mutation of the STM1082 gene down-regulates the expression of multiple genes involved in purine biosynthesis in N-minimal medium, which mimics the conditions inside macrophages [37,38,48,49]. Purines are components of genetic material and play a central role in cellular metabolism. They are incorporated into coenzymes (e.g. FAD, NAD⁺, NADP⁺, and coenzyme A), serve as signaling molecules (e.g. ATP, cAMP, and cGMP), act as phosphate donors, and function as the energy currency of cells (ATP and GTP) [43,75]. In Salmonella, the pur genes encode the enzymes necessary for purine biosynthesis. These genes are scattered throughout the entire Salmonella genome in the form of small operons or individual genes. Previous studies demonstrate that knocking out the purE gene, a key component of the purine biosynthetic pathway, attenuates Salmonella virulence in cattle following oral infection and reduces liver/spleen colonization in BALB/c mice following intravenous injection [54,55]. Our study further reveal that i.p. infection of BALB/c mice with the purE mutant leads to significantly reduced bacterial counts in systemic tissues and increased mouse body weight. Furthermore, we have demonstrated that the replication ability of the purE mutant within mouse macrophages was significantly decreased. These results confirm the essential role of purE and the associated purine biosynthesis pathway in enabling Salmonella to replicate within macrophages and to establish systemic infections in mice. Considering the crucial role of purine biosynthesis in the intracellular replication of Salmonella and its ability to cause systemic infection, it is highly probable that STM1082 facilitates Salmonella‘s intracellular replication and systemic infection by up-regulating purine biosynthesis.

In summary, we have characterized that STM1082, a putative AraC-type transcription regulator, as a critical virulence factor of Salmonella. STM1082 enhances pathogenicity by two key mechanisms: i) promoting intestinal invasion and intestinal growth (likely via upregulating 1,2-propanediol and ethanolamine utilization), and ii) augmenting replication within macrophages and systemic infection (possibly through upregulation of purine biosynthesis genes). Notably, STM1082 is conserved across major human-pathogenic Salmonella serovars, including S. Typhimurium, S. Typhi, S. Paratyphi, and S. Enteritidis. Considering that mutation of the STM1082 gene significantly reduces Salmonella’s invasion and replication capabilities, this gene could serve as a potential target for controlling Salmonella infection.

Supplementary Material

20250714 figs1.tif

KVIR_A_2554297_SM2678.tif^{(191.1KB, tif)}

20250714 STM1082_Supplemental data.docx

KVIR_A_2554297_SM2677.docx^{(129.2KB, docx)}

Acknowledgements

LJ designed the research; SM, XW, YS, HG, and JM performed the research; WL provided technical support and insights; SM and WL analyzed the data; and LJ and SM wrote the manuscript.

Funding Statement

This work was supported by the National Natural Science Foundation of China (NSFC) [Grant No. 32170110], the Laboratory Major Project of Tianjin in 2024 [Grant No. 24ZXZSSS00140], the Natural Science Foundation of Tianjin [Grant No. 22JCYBJC01060], and the Innovation Training Program for College Students in Henan Province [Grant No. S202510464084].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The RNA-seq data generated in this research have been submitted to the NCBI Sequence Read Archive (SRA) database. The accession number for these data is PRJNA1247279. All other data related to this study can be found in the main text and supplementary materials. Source data of this study are openly available in Zenodo (https://doi.org/10.5281/zenodo.15869083).

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2025.2554297

References

[1].Besser JM. Salmonella epidemiology: a whirlwind of change. Food microbiol. 2018;71:55–20. doi: 10.1016/j.fm.2017.08.018 [DOI] [PubMed] [Google Scholar]
[2].Fàbrega A, Vila J. Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev. 2013;26(2):308–341. doi: 10.1128/cmr.00066-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
[3].Ohl ME, Miller SI. Salmonella: a model for bacterial pathogenesis. Annu Rev Med. 2001;52(1):259–274. doi: 10.1146/annurev.med.52.1.259 [DOI] [PubMed] [Google Scholar]
[4].Han J, Aljahdali N, Zhao S, et al. Infection biology of Salmonella enterica. ecosal PLus. 2024;12(1):eesp00012023. doi: 10.1128/ecosalplus.esp-0001-2023 [DOI] [PMC free article] [PubMed] [Google Scholar]
[5].Fierer J. Invasive non-typhoidal salmonella (iNTS) infections. Clin Infect Dis. 2022;75(4):732–738. doi: 10.1093/cid/ciac035 [DOI] [PubMed] [Google Scholar]
[6].Karlinsey JE, Stepien TA, Mayho M, et al. Genome-wide analysis of Salmonella enterica serovar Typhi in humanized mice reveals key virulence features. Cell Host Microbe. 2019;26(3):426–434.e6. doi: 10.1016/j.chom.2019.08.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
[7].Kenney LJ. The role of acid stress in Salmonella pathogenesis. Curr Opin microbiol. 2019;47:45–51. doi: 10.1016/j.mib.2018.11.006 [DOI] [PubMed] [Google Scholar]
[8].Mabbott NA, Donaldson DS, Ohno H, et al. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 2013;6(4):666–677. doi: 10.1038/mi.2013.30 [DOI] [PMC free article] [PubMed] [Google Scholar]
[9].Ruby T, McLaughlin L, Gopinath S, et al. Salmonella‘s long-term relationship with its host. FEMS Microbiol Rev. 2012;36(3):600–615. doi: 10.1111/j.1574-6976.2012.00332.x [DOI] [PubMed] [Google Scholar]
[10].Salcedo SP, Noursadeghi M, Cohen J, et al. Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo. Cell microbiol. 2001;3(9):587–597. doi: 10.1046/j.1462-5822.2001.00137.x [DOI] [PubMed] [Google Scholar]
[11].Mastroeni P, Grant AJ. Spread of Salmonella enterica in the body during systemic infection: unravelling host and pathogen determinants. Expert Rev Mol Med. 2011;13:e12. doi: 10.1017/s1462399411001840 [DOI] [PubMed] [Google Scholar]
[12].Jennings E, Thurston TLM, Holden DW. Salmonella SPI-2 type III secretion system effectors: molecular mechanisms and physiological consequences. Cell Host Microbe. 2017;22(2):217–231. doi: 10.1016/j.chom.2017.07.009 [DOI] [PubMed] [Google Scholar]
[13].Arpaia N, Godec J, Lau L, et al. Tlr signaling is required for Salmonella typhimurium virulence. Cell. 2011;144(5):675–688. doi: 10.1016/j.cell.2011.01.031 [DOI] [PMC free article] [PubMed] [Google Scholar]
[14].Watson KG, Holden DW. Dynamics of growth and dissemination of Salmonella in vivo. Cell microbiol. 2010;12(10):1389–1397. doi: 10.1111/j.1462-5822.2010.01511.x [DOI] [PubMed] [Google Scholar]
[15].Zhou D, Galán J. Salmonella entry into host cells: the work in concert of type III secreted effector proteins. Microbes Infect. 2001;3(14–15):1293–1298. doi: 10.1016/s1286-4579(01)01489-7 [DOI] [PubMed] [Google Scholar]
[16].Li W, Ren Q, Ni T, et al. Strategies adopted by Salmonella to survive in host: a review. Arch microbiol. 2023;205(12):362. doi: 10.1007/s00203-023-03702-w [DOI] [PubMed] [Google Scholar]
[17].Figueira R, Holden DW. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiol (read). 2012;158(Pt 5):1147–1161. doi: 10.1099/mic.0.058115-0 [DOI] [PubMed] [Google Scholar]
[18].Lou L, Zhang P, Piao R, et al. Salmonella pathogenicity island 1 (SPI-1) and its complex regulatory network. Front Cell Infect microbiol. 2019;9:270. doi: 10.3389/fcimb.2019.00270 [DOI] [PMC free article] [PubMed] [Google Scholar]
[19].Wang X, Yang B, Ma S, et al. Lactate promotes Salmonella intracellular replication and systemic infection via driving macrophage M2 polarization. Microbiol spectr. 2023;11(6):e0225323. doi: 10.1128/spectrum.02253-23 [DOI] [PMC free article] [PubMed] [Google Scholar]
[20].Jiang L, Wang P, Song X, et al. Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence. Nat Commun. 2021;12(1):879. doi: 10.1038/s41467-021-21186-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
[21].Wang X, Sun Y, Guo H, et al. Xylose utilization promotes Salmonella replication within macrophages and systemic infection in mice. Virulence. 2024;15(1):2435381. doi: 10.1080/21505594.2024.2435381 [DOI] [PMC free article] [PubMed] [Google Scholar]
[22].Ma S, Yang B, Sun Y, et al. Salmonella exploits host- and bacterial-derived β-alanine for replication inside host macrophages. Elife. 2025;13:RP103714. doi: 10.7554/eLife.103714 [DOI] [PMC free article] [PubMed] [Google Scholar]
[23].Hassett DJ, Cohen MS. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. Faseb J. 1989;3(14):2574–2582. doi: 10.1096/fasebj.3.14.2556311 [DOI] [PubMed] [Google Scholar]
[24].Pardo-Esté C, Hidalgo AA, Aguirre C, et al. The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium. PLOS ONE. 2018;13(9):e0203497. doi: 10.1371/journal.pone.0203497 [DOI] [PMC free article] [PubMed] [Google Scholar]
[25].Bourret TJ, Liu L, Shaw JA, et al. Magnesium homeostasis protects Salmonella against nitrooxidative stress. Sci Rep. 2017;7(1):15083. doi: 10.1038/s41598-017-15445-y [DOI] [PMC free article] [PubMed] [Google Scholar]
[26].Li W, Ma S, Yan X, et al. The LysR-type transcription regulator YhjC promotes the systemic infection of Salmonella typhimurium in mice. Int J Mol Sci. 2023;24(2):1302. doi: 10.3390/ijms24021302 [DOI] [PMC free article] [PubMed] [Google Scholar]
[27].Guadarrama C, Villaseñor T, Calva E. The subtleties and contrasts of the LeuO regulator in Salmonella Typhi: implications in the immune response. Front Immunol. 2014;5:581. doi: 10.3389/fimmu.2014.00581 [DOI] [PMC free article] [PubMed] [Google Scholar]
[28].Guo J, He X, Bai Y, et al. Virulence factors of Salmonella Typhi: interplay between the bacteria and host macrophages. Arch microbiol. 2025;207(4):89. doi: 10.1007/s00203-025-04297-0 [DOI] [PubMed] [Google Scholar]
[29].Pardo-Esté C, Urbina F, Aviles N, et al. The Arcb kinase sensor participates in the phagocyte-mediated stress response in Salmonella typhimurium. Front microbiol. 2025;16:1541797. doi: 10.3389/fmicb.2025.1541797 [DOI] [PMC free article] [PubMed] [Google Scholar]
[30].Carey J. Affinity, specificity, and cooperativity of DNA binding by bacterial gene regulatory proteins. Int J Mol Sci. 2022;23(1):562. doi: 10.3390/ijms23010562 [DOI] [PMC free article] [PubMed] [Google Scholar]
[31].Feng X, Walthers D, Oropeza R, et al. The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2. Mol microbiol. 2004;54(3):823–835. doi: 10.1111/j.1365-2958.2004.04317.x [DOI] [PubMed] [Google Scholar]
[32].Jiang X, Li X, Sun S, et al. The transcriptional regulator VarN contributes to Salmonella Typhimurium growth in macrophages and virulence in mice. Res microbiol. 2018;169(4–5):214–221. doi: 10.1016/j.resmic.2018.03.003 [DOI] [PubMed] [Google Scholar]
[33].Jiang L, Wang P, Li X, et al. Pagr mediates the precise regulation of salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in salmonella Typhimurium. Cell microbiol. 2020;22(2):e13125. doi: 10.1111/cmi.13125 [DOI] [PubMed] [Google Scholar]
[34].Srikumar S, Kröger C, Hébrard M, et al. Rna-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium. PLoS Pathog. 2015;11(11):e1005262. doi: 10.1371/journal.ppat.1005262 [DOI] [PMC free article] [PubMed] [Google Scholar]
[35].Cardenal-Muñoz E, Ramos-Morales F, Chakravortty D. Analysis of the expression, secretion and translocation of the salmonella enterica type III secretion system effector SteA. PLOS ONE. 2011;6(10):e26930. doi: 10.1371/journal.pone.0026930 [DOI] [PMC free article] [PubMed] [Google Scholar]
[36].Lundberg U, Vinatzer U, Berdnik D, et al. Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes. J Bacteriol. 1999;181(11):3433–3437. doi: 10.1128/jb.181.11.3433-3437.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]
[37].Bustamante VH, Martínez LC, Santana FJ, et al. Hild-mediated transcriptional cross-talk between SPI-1 and SPI-2. Proc Natl Acad Sci USA. 2008;105(38):14591–14596. doi: 10.1073/pnas.0801205105 [DOI] [PMC free article] [PubMed] [Google Scholar]
[38].Kröger C, Colgan A, Srikumar S, et al. An infection-relevant transcriptomic compendium for Salmonella enterica serovar Typhimurium. Cell Host Microbe. 2013;14(6):683–695. doi: 10.1016/j.chom.2013.11.010 [DOI] [PubMed] [Google Scholar]
[39].Jeter RM. Cobalamin-dependent 1,2-propanediol utilization by Salmonella Typhimurium. J Gen microbiol. 1990;136(5):887–896. doi: 10.1099/00221287-136-5-887 [DOI] [PubMed] [Google Scholar]
[40].Chang GW, Chang JT. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature. 1975;254(5496):150–151. doi: 10.1038/254150a0 [DOI] [PubMed] [Google Scholar]
[41].Roof DM, Roth JR. Ethanolamine utilization in Salmonella Typhimurium. J Bacteriol. 1988;170(9):3855–3863. doi: 10.1128/jb.170.9.3855-3863.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
[42].Roof DM, Roth JR. Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella Typhimurium. J Bacteriol. 1989;171(6):3316–3323. doi: 10.1128/jb.171.6.3316-3323.1989 [DOI] [PMC free article] [PubMed] [Google Scholar]
[43].Pareek V, Pedley AM, Benkovic SJ. Human de novo purine biosynthesis. Crit Rev Biochem Mol Biol. 2021;56(1):1–16. doi: 10.1080/10409238.2020.1832438 [DOI] [PMC free article] [PubMed] [Google Scholar]
[44].Jiang L, Feng L, Yang B, et al. Signal transduction pathway mediated by the novel regulator LoiA for low oxygen tension induced Salmonella Typhimurium invasion. PLoS Pathog. 2017;13(6):e1006429. doi: 10.1371/journal.ppat.1006429 [DOI] [PMC free article] [PubMed] [Google Scholar]
[45].Liang H, Li L, Dong Z, et al. The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production. J Bacteriol. 2008;190(18):6217–6227. doi: 10.1128/jb.00428-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
[46].Faber F, Thiennimitr P, Spiga L, et al. Respiration of microbiota-derived 1,2-propanediol drives Salmonella expansion during colitis. PLoS Pathog. 2017;13(1):e1006129. doi: 10.1371/journal.ppat.1006129 [DOI] [PMC free article] [PubMed] [Google Scholar]
[47].Anderson CJ, Clark DE, Adli M, et al. Ethanolamine signaling promotes Salmonella niche recognition and adaptation during infection. PLoS Pathog. 2015;11(11):e1005278. doi: 10.1371/journal.ppat.1005278 [DOI] [PMC free article] [PubMed] [Google Scholar]
[48].Deiwick J, Nikolaus T, Erdogan S, et al. Environmental regulation of salmonella pathogenicity island 2 gene expression. Mol microbiol. 1999;31(6):1759–1773. doi: 10.1046/j.1365-2958.1999.01312.x [DOI] [PubMed] [Google Scholar]
[49].Miao EA, Miller SI. A conserved amino acid sequence directing intracellular type III secretion by Salmonella Typhimurium. Proc Natl Acad Sci USA. 2000;97(13):7539–7544. doi: 10.1073/pnas.97.13.7539 [DOI] [PMC free article] [PubMed] [Google Scholar]
[50].Duffy SC, Lupien A, Elhaji Y, et al. Establishment of persistent enteric mycobacterial infection following streptomycin pre-treatment. Gut Pathog. 2023;15(1):46. doi: 10.1186/s13099-023-00573-w [DOI] [PMC free article] [PubMed] [Google Scholar]
[51].Pandeya A, Zhang Y, Cui J, et al. Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection. Microbiol Res. 2023;275:127460. doi: 10.1016/j.micres.2023.127460 [DOI] [PMC free article] [PubMed] [Google Scholar]
[52].Silva AF, Matos MP, Ralph MT, et al. Comparison of immunomodulatory properties of mannose-binding lectins from Canavalia brasiliensis and Cratylia argentea in a mice model of Salmonella infection. Int immunopharmacol. 2016;31:233–238. doi: 10.1016/j.intimp.2015.12.036 [DOI] [PubMed] [Google Scholar]
[53].Lerminiaux NA, MacKenzie KD, Cameron ADS. Salmonella pathogenicity island 1 (SPI-1): the evolution and stabilization of a core genomic type three secretion system. Microorganisms. 2020;8(4):576. doi: 10.3390/microorganisms8040576 [DOI] [PMC free article] [PubMed] [Google Scholar]
[54].Villarreal-Ramos B, Manser JM, Collins RA, et al. Susceptibility of calves to challenge with Salmonella typhimurium 4/74 and derivatives harbouring mutations in htra or pure. Microbiol (read). 2000;146(Pt 11):2775–2783. doi: 10.1099/00221287-146-11-2775 [DOI] [PubMed] [Google Scholar]
[55].O’Callaghan D, Maskell D, Liew FY, et al. Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice. Infect Immun. 1988;56(2):419–423. doi: 10.1128/iai.56.2.419-423.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
[56].Chowdhury R, Pavinski Bitar PD, Adams MC, et al. AraC-type regulators HilC and RtsA are directly controlled by an intestinal fatty acid to regulate Salmonella invasion. Mol microbiol. 2021;116(6):1464–1475. doi: 10.1111/mmi.14835 [DOI] [PMC free article] [PubMed] [Google Scholar]
[57].Bailey AM, Ivens A, Kingsley R, et al. Rama, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol. 2010;192(6):1607–1616. doi: 10.1128/jb.01517-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
[58].Linehan SA, Rytkönen A, Yu XJ, et al. Slya regulates function of salmonella pathogenicity island 2 (SPI-2) and expression of SPI-2-associated genes. Infect Immun. 2005;73(7):4354–4362. doi: 10.1128/iai.73.7.4354-4362.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
[59].Garmendia J, Beuzón CR, Ruiz-Albert J, et al. The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella Typhimurium SPI-2 type III secretion system. Microbiol (read). 2003;149(Pt 9):2385–2396. doi: 10.1099/mic.0.26397-0 [DOI] [PubMed] [Google Scholar]
[60].Gallegos MT, Schleif R, Bairoch A, et al. Arac/xyls family of transcriptional regulators. Microbiol Mol Biol Rev. 1997;61(4):393–410. doi: 10.1128/mmbr.61.4.393-410.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
[61].Darwin KH, Miller VL. Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. Embo J. 2001;20(8):1850–1862. doi: 10.1093/emboj/20.8.1850 [DOI] [PMC free article] [PubMed] [Google Scholar]
[62].Obradors N, Badía J, Baldomà L, et al. Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium. J Bacteriol. 1988;170(5):2159–2162. doi: 10.1128/jb.170.5.2159-2162.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
[63].Bobik TA, Xu Y, Jeter RM, et al. Propanediol utilization genes (pdu) of Salmonella Typhimurium: three genes for the propanediol dehydratase. J Bacteriol. 1997;179(21):6633–6639. doi: 10.1128/jb.179.21.6633-6639.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
[64].Larson TJ, Ehrmann M, Boos W. Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon. J Biol Chem. 1983;258(9):5428–5432. doi: 10.1016/S0021-9258(20)81908-5 [DOI] [PubMed] [Google Scholar]
[65].Fung CK, Proulx P. Metabolism of phosphoglycerides in E. coli. 3. The presence of phospholipase A. Can J biochem. 1969;47(3):371–373. doi: 10.1139/o69-055 [DOI] [PubMed] [Google Scholar]
[66].Cotton PB. Non-dietary lipid in the intestinal lumen. Gut. 1972;13(9):675–681. doi: 10.1136/gut.13.9.675 [DOI] [PMC free article] [PubMed] [Google Scholar]
[67].Brinsmade SR, Paldon T, Escalante-Semerena JC. Minimal functions and physiological conditions required for growth of Salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol. 2005;187(23):8039–8046. doi: 10.1128/jb.187.23.8039-8046.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
[68].Cheng S, Sinha S, Fan C, et al. Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella. J Bacteriol. 2011;193(6):1385–1392. doi: 10.1128/jb.01473-10 [DOI] [PMC free article] [PubMed] [Google Scholar]
[69].Stewart KL, Stewart AM, Bobik TA, et al. Prokaryotic organelles: bacterial microcompartments in E. coli and Salmonella. ecosal PLus. 2020;9(1): doi: 10.1128/ecosalplus.ESP-0025-2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
[70].Chowdhury C, Chun S, Sawaya MR, et al. The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene. Mol microbiol. 2016;101(5):770–783. doi: 10.1111/mmi.13423 [DOI] [PMC free article] [PubMed] [Google Scholar]
[71].Cheng S, Fan C, Sinha S, et al. The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica. PLOS ONE. 2012;7(10):e47144. doi: 10.1371/journal.pone.0047144 [DOI] [PMC free article] [PubMed] [Google Scholar]
[72].Penrod JT, Roth JR. Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol. 2006;188(8):2865–2874. doi: 10.1128/jb.188.8.2865-2874.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
[73].Fattinger SA, Sellin ME, Hardt WD. Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire. Curr Opin microbiol. 2021;64:9–18. doi: 10.1016/j.mib.2021.08.007 [DOI] [PubMed] [Google Scholar]
[74].Thiennimitr P, Winter SE, Winter MG, et al. Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota. Proc Natl Acad Sci USA. 2011;108(42):17480–17485. doi: 10.1073/pnas.1107857108 [DOI] [PMC free article] [PubMed] [Google Scholar]
[75].Rolfes RJ. Regulation of purine nucleotide biosynthesis: in yeast and beyond. Biochem Soc Trans. 2006;34(Pt 5):786–790. doi: 10.1042/bst0340786 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

20250714 figs1.tif

KVIR_A_2554297_SM2678.tif^{(191.1KB, tif)}

20250714 STM1082_Supplemental data.docx

KVIR_A_2554297_SM2677.docx^{(129.2KB, docx)}

Data Availability Statement

[cit0001] [1].Besser JM. Salmonella epidemiology: a whirlwind of change. Food microbiol. 2018;71:55–20. doi: 10.1016/j.fm.2017.08.018 [DOI] [PubMed] [Google Scholar]

[cit0002] [2].Fàbrega A, Vila J. Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev. 2013;26(2):308–341. doi: 10.1128/cmr.00066-12 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0003] [3].Ohl ME, Miller SI. Salmonella: a model for bacterial pathogenesis. Annu Rev Med. 2001;52(1):259–274. doi: 10.1146/annurev.med.52.1.259 [DOI] [PubMed] [Google Scholar]

[cit0004] [4].Han J, Aljahdali N, Zhao S, et al. Infection biology of Salmonella enterica. ecosal PLus. 2024;12(1):eesp00012023. doi: 10.1128/ecosalplus.esp-0001-2023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0005] [5].Fierer J. Invasive non-typhoidal salmonella (iNTS) infections. Clin Infect Dis. 2022;75(4):732–738. doi: 10.1093/cid/ciac035 [DOI] [PubMed] [Google Scholar]

[cit0006] [6].Karlinsey JE, Stepien TA, Mayho M, et al. Genome-wide analysis of Salmonella enterica serovar Typhi in humanized mice reveals key virulence features. Cell Host Microbe. 2019;26(3):426–434.e6. doi: 10.1016/j.chom.2019.08.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0007] [7].Kenney LJ. The role of acid stress in Salmonella pathogenesis. Curr Opin microbiol. 2019;47:45–51. doi: 10.1016/j.mib.2018.11.006 [DOI] [PubMed] [Google Scholar]

[cit0008] [8].Mabbott NA, Donaldson DS, Ohno H, et al. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol. 2013;6(4):666–677. doi: 10.1038/mi.2013.30 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0009] [9].Ruby T, McLaughlin L, Gopinath S, et al. Salmonella‘s long-term relationship with its host. FEMS Microbiol Rev. 2012;36(3):600–615. doi: 10.1111/j.1574-6976.2012.00332.x [DOI] [PubMed] [Google Scholar]

[cit0010] [10].Salcedo SP, Noursadeghi M, Cohen J, et al. Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo. Cell microbiol. 2001;3(9):587–597. doi: 10.1046/j.1462-5822.2001.00137.x [DOI] [PubMed] [Google Scholar]

[cit0011] [11].Mastroeni P, Grant AJ. Spread of Salmonella enterica in the body during systemic infection: unravelling host and pathogen determinants. Expert Rev Mol Med. 2011;13:e12. doi: 10.1017/s1462399411001840 [DOI] [PubMed] [Google Scholar]

[cit0012] [12].Jennings E, Thurston TLM, Holden DW. Salmonella SPI-2 type III secretion system effectors: molecular mechanisms and physiological consequences. Cell Host Microbe. 2017;22(2):217–231. doi: 10.1016/j.chom.2017.07.009 [DOI] [PubMed] [Google Scholar]

[cit0013] [13].Arpaia N, Godec J, Lau L, et al. Tlr signaling is required for Salmonella typhimurium virulence. Cell. 2011;144(5):675–688. doi: 10.1016/j.cell.2011.01.031 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0014] [14].Watson KG, Holden DW. Dynamics of growth and dissemination of Salmonella in vivo. Cell microbiol. 2010;12(10):1389–1397. doi: 10.1111/j.1462-5822.2010.01511.x [DOI] [PubMed] [Google Scholar]

[cit0015] [15].Zhou D, Galán J. Salmonella entry into host cells: the work in concert of type III secreted effector proteins. Microbes Infect. 2001;3(14–15):1293–1298. doi: 10.1016/s1286-4579(01)01489-7 [DOI] [PubMed] [Google Scholar]

[cit0016] [16].Li W, Ren Q, Ni T, et al. Strategies adopted by Salmonella to survive in host: a review. Arch microbiol. 2023;205(12):362. doi: 10.1007/s00203-023-03702-w [DOI] [PubMed] [Google Scholar]

[cit0017] [17].Figueira R, Holden DW. Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiol (read). 2012;158(Pt 5):1147–1161. doi: 10.1099/mic.0.058115-0 [DOI] [PubMed] [Google Scholar]

[cit0018] [18].Lou L, Zhang P, Piao R, et al. Salmonella pathogenicity island 1 (SPI-1) and its complex regulatory network. Front Cell Infect microbiol. 2019;9:270. doi: 10.3389/fcimb.2019.00270 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0019] [19].Wang X, Yang B, Ma S, et al. Lactate promotes Salmonella intracellular replication and systemic infection via driving macrophage M2 polarization. Microbiol spectr. 2023;11(6):e0225323. doi: 10.1128/spectrum.02253-23 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0020] [20].Jiang L, Wang P, Song X, et al. Salmonella Typhimurium reprograms macrophage metabolism via T3SS effector SopE2 to promote intracellular replication and virulence. Nat Commun. 2021;12(1):879. doi: 10.1038/s41467-021-21186-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0021] [21].Wang X, Sun Y, Guo H, et al. Xylose utilization promotes Salmonella replication within macrophages and systemic infection in mice. Virulence. 2024;15(1):2435381. doi: 10.1080/21505594.2024.2435381 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0022] [22].Ma S, Yang B, Sun Y, et al. Salmonella exploits host- and bacterial-derived β-alanine for replication inside host macrophages. Elife. 2025;13:RP103714. doi: 10.7554/eLife.103714 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0023] [23].Hassett DJ, Cohen MS. Bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells. Faseb J. 1989;3(14):2574–2582. doi: 10.1096/fasebj.3.14.2556311 [DOI] [PubMed] [Google Scholar]

[cit0024] [24].Pardo-Esté C, Hidalgo AA, Aguirre C, et al. The ArcAB two-component regulatory system promotes resistance to reactive oxygen species and systemic infection by Salmonella Typhimurium. PLOS ONE. 2018;13(9):e0203497. doi: 10.1371/journal.pone.0203497 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0025] [25].Bourret TJ, Liu L, Shaw JA, et al. Magnesium homeostasis protects Salmonella against nitrooxidative stress. Sci Rep. 2017;7(1):15083. doi: 10.1038/s41598-017-15445-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0026] [26].Li W, Ma S, Yan X, et al. The LysR-type transcription regulator YhjC promotes the systemic infection of Salmonella typhimurium in mice. Int J Mol Sci. 2023;24(2):1302. doi: 10.3390/ijms24021302 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0027] [27].Guadarrama C, Villaseñor T, Calva E. The subtleties and contrasts of the LeuO regulator in Salmonella Typhi: implications in the immune response. Front Immunol. 2014;5:581. doi: 10.3389/fimmu.2014.00581 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0028] [28].Guo J, He X, Bai Y, et al. Virulence factors of Salmonella Typhi: interplay between the bacteria and host macrophages. Arch microbiol. 2025;207(4):89. doi: 10.1007/s00203-025-04297-0 [DOI] [PubMed] [Google Scholar]

[cit0029] [29].Pardo-Esté C, Urbina F, Aviles N, et al. The Arcb kinase sensor participates in the phagocyte-mediated stress response in Salmonella typhimurium. Front microbiol. 2025;16:1541797. doi: 10.3389/fmicb.2025.1541797 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0030] [30].Carey J. Affinity, specificity, and cooperativity of DNA binding by bacterial gene regulatory proteins. Int J Mol Sci. 2022;23(1):562. doi: 10.3390/ijms23010562 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0031] [31].Feng X, Walthers D, Oropeza R, et al. The response regulator SsrB activates transcription and binds to a region overlapping OmpR binding sites at Salmonella pathogenicity island 2. Mol microbiol. 2004;54(3):823–835. doi: 10.1111/j.1365-2958.2004.04317.x [DOI] [PubMed] [Google Scholar]

[cit0032] [32].Jiang X, Li X, Sun S, et al. The transcriptional regulator VarN contributes to Salmonella Typhimurium growth in macrophages and virulence in mice. Res microbiol. 2018;169(4–5):214–221. doi: 10.1016/j.resmic.2018.03.003 [DOI] [PubMed] [Google Scholar]

[cit0033] [33].Jiang L, Wang P, Li X, et al. Pagr mediates the precise regulation of salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in salmonella Typhimurium. Cell microbiol. 2020;22(2):e13125. doi: 10.1111/cmi.13125 [DOI] [PubMed] [Google Scholar]

[cit0034] [34].Srikumar S, Kröger C, Hébrard M, et al. Rna-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium. PLoS Pathog. 2015;11(11):e1005262. doi: 10.1371/journal.ppat.1005262 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0035] [35].Cardenal-Muñoz E, Ramos-Morales F, Chakravortty D. Analysis of the expression, secretion and translocation of the salmonella enterica type III secretion system effector SteA. PLOS ONE. 2011;6(10):e26930. doi: 10.1371/journal.pone.0026930 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0036] [36].Lundberg U, Vinatzer U, Berdnik D, et al. Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes. J Bacteriol. 1999;181(11):3433–3437. doi: 10.1128/jb.181.11.3433-3437.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0037] [37].Bustamante VH, Martínez LC, Santana FJ, et al. Hild-mediated transcriptional cross-talk between SPI-1 and SPI-2. Proc Natl Acad Sci USA. 2008;105(38):14591–14596. doi: 10.1073/pnas.0801205105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0038] [38].Kröger C, Colgan A, Srikumar S, et al. An infection-relevant transcriptomic compendium for Salmonella enterica serovar Typhimurium. Cell Host Microbe. 2013;14(6):683–695. doi: 10.1016/j.chom.2013.11.010 [DOI] [PubMed] [Google Scholar]

[cit0039] [39].Jeter RM. Cobalamin-dependent 1,2-propanediol utilization by Salmonella Typhimurium. J Gen microbiol. 1990;136(5):887–896. doi: 10.1099/00221287-136-5-887 [DOI] [PubMed] [Google Scholar]

[cit0040] [40].Chang GW, Chang JT. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature. 1975;254(5496):150–151. doi: 10.1038/254150a0 [DOI] [PubMed] [Google Scholar]

[cit0041] [41].Roof DM, Roth JR. Ethanolamine utilization in Salmonella Typhimurium. J Bacteriol. 1988;170(9):3855–3863. doi: 10.1128/jb.170.9.3855-3863.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0042] [42].Roof DM, Roth JR. Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella Typhimurium. J Bacteriol. 1989;171(6):3316–3323. doi: 10.1128/jb.171.6.3316-3323.1989 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0043] [43].Pareek V, Pedley AM, Benkovic SJ. Human de novo purine biosynthesis. Crit Rev Biochem Mol Biol. 2021;56(1):1–16. doi: 10.1080/10409238.2020.1832438 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0044] [44].Jiang L, Feng L, Yang B, et al. Signal transduction pathway mediated by the novel regulator LoiA for low oxygen tension induced Salmonella Typhimurium invasion. PLoS Pathog. 2017;13(6):e1006429. doi: 10.1371/journal.ppat.1006429 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0045] [45].Liang H, Li L, Dong Z, et al. The YebC family protein PA0964 negatively regulates the Pseudomonas aeruginosa quinolone signal system and pyocyanin production. J Bacteriol. 2008;190(18):6217–6227. doi: 10.1128/jb.00428-08 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0046] [46].Faber F, Thiennimitr P, Spiga L, et al. Respiration of microbiota-derived 1,2-propanediol drives Salmonella expansion during colitis. PLoS Pathog. 2017;13(1):e1006129. doi: 10.1371/journal.ppat.1006129 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0047] [47].Anderson CJ, Clark DE, Adli M, et al. Ethanolamine signaling promotes Salmonella niche recognition and adaptation during infection. PLoS Pathog. 2015;11(11):e1005278. doi: 10.1371/journal.ppat.1005278 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0048] [48].Deiwick J, Nikolaus T, Erdogan S, et al. Environmental regulation of salmonella pathogenicity island 2 gene expression. Mol microbiol. 1999;31(6):1759–1773. doi: 10.1046/j.1365-2958.1999.01312.x [DOI] [PubMed] [Google Scholar]

[cit0049] [49].Miao EA, Miller SI. A conserved amino acid sequence directing intracellular type III secretion by Salmonella Typhimurium. Proc Natl Acad Sci USA. 2000;97(13):7539–7544. doi: 10.1073/pnas.97.13.7539 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0050] [50].Duffy SC, Lupien A, Elhaji Y, et al. Establishment of persistent enteric mycobacterial infection following streptomycin pre-treatment. Gut Pathog. 2023;15(1):46. doi: 10.1186/s13099-023-00573-w [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0051] [51].Pandeya A, Zhang Y, Cui J, et al. Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection. Microbiol Res. 2023;275:127460. doi: 10.1016/j.micres.2023.127460 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0052] [52].Silva AF, Matos MP, Ralph MT, et al. Comparison of immunomodulatory properties of mannose-binding lectins from Canavalia brasiliensis and Cratylia argentea in a mice model of Salmonella infection. Int immunopharmacol. 2016;31:233–238. doi: 10.1016/j.intimp.2015.12.036 [DOI] [PubMed] [Google Scholar]

[cit0053] [53].Lerminiaux NA, MacKenzie KD, Cameron ADS. Salmonella pathogenicity island 1 (SPI-1): the evolution and stabilization of a core genomic type three secretion system. Microorganisms. 2020;8(4):576. doi: 10.3390/microorganisms8040576 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0054] [54].Villarreal-Ramos B, Manser JM, Collins RA, et al. Susceptibility of calves to challenge with Salmonella typhimurium 4/74 and derivatives harbouring mutations in htra or pure. Microbiol (read). 2000;146(Pt 11):2775–2783. doi: 10.1099/00221287-146-11-2775 [DOI] [PubMed] [Google Scholar]

[cit0055] [55].O’Callaghan D, Maskell D, Liew FY, et al. Characterization of aromatic- and purine-dependent Salmonella typhimurium: attention, persistence, and ability to induce protective immunity in BALB/c mice. Infect Immun. 1988;56(2):419–423. doi: 10.1128/iai.56.2.419-423.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0056] [56].Chowdhury R, Pavinski Bitar PD, Adams MC, et al. AraC-type regulators HilC and RtsA are directly controlled by an intestinal fatty acid to regulate Salmonella invasion. Mol microbiol. 2021;116(6):1464–1475. doi: 10.1111/mmi.14835 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0057] [57].Bailey AM, Ivens A, Kingsley R, et al. Rama, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol. 2010;192(6):1607–1616. doi: 10.1128/jb.01517-09 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0058] [58].Linehan SA, Rytkönen A, Yu XJ, et al. Slya regulates function of salmonella pathogenicity island 2 (SPI-2) and expression of SPI-2-associated genes. Infect Immun. 2005;73(7):4354–4362. doi: 10.1128/iai.73.7.4354-4362.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0059] [59].Garmendia J, Beuzón CR, Ruiz-Albert J, et al. The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella Typhimurium SPI-2 type III secretion system. Microbiol (read). 2003;149(Pt 9):2385–2396. doi: 10.1099/mic.0.26397-0 [DOI] [PubMed] [Google Scholar]

[cit0060] [60].Gallegos MT, Schleif R, Bairoch A, et al. Arac/xyls family of transcriptional regulators. Microbiol Mol Biol Rev. 1997;61(4):393–410. doi: 10.1128/mmbr.61.4.393-410.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0061] [61].Darwin KH, Miller VL. Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. Embo J. 2001;20(8):1850–1862. doi: 10.1093/emboj/20.8.1850 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0062] [62].Obradors N, Badía J, Baldomà L, et al. Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium. J Bacteriol. 1988;170(5):2159–2162. doi: 10.1128/jb.170.5.2159-2162.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0063] [63].Bobik TA, Xu Y, Jeter RM, et al. Propanediol utilization genes (pdu) of Salmonella Typhimurium: three genes for the propanediol dehydratase. J Bacteriol. 1997;179(21):6633–6639. doi: 10.1128/jb.179.21.6633-6639.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0064] [64].Larson TJ, Ehrmann M, Boos W. Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon. J Biol Chem. 1983;258(9):5428–5432. doi: 10.1016/S0021-9258(20)81908-5 [DOI] [PubMed] [Google Scholar]

[cit0065] [65].Fung CK, Proulx P. Metabolism of phosphoglycerides in E. coli. 3. The presence of phospholipase A. Can J biochem. 1969;47(3):371–373. doi: 10.1139/o69-055 [DOI] [PubMed] [Google Scholar]

[cit0066] [66].Cotton PB. Non-dietary lipid in the intestinal lumen. Gut. 1972;13(9):675–681. doi: 10.1136/gut.13.9.675 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0067] [67].Brinsmade SR, Paldon T, Escalante-Semerena JC. Minimal functions and physiological conditions required for growth of Salmonella enterica on ethanolamine in the absence of the metabolosome. J Bacteriol. 2005;187(23):8039–8046. doi: 10.1128/jb.187.23.8039-8046.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0068] [68].Cheng S, Sinha S, Fan C, et al. Genetic analysis of the protein shell of the microcompartments involved in coenzyme B12-dependent 1,2-propanediol degradation by Salmonella. J Bacteriol. 2011;193(6):1385–1392. doi: 10.1128/jb.01473-10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0069] [69].Stewart KL, Stewart AM, Bobik TA, et al. Prokaryotic organelles: bacterial microcompartments in E. coli and Salmonella. ecosal PLus. 2020;9(1): doi: 10.1128/ecosalplus.ESP-0025-2019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0070] [70].Chowdhury C, Chun S, Sawaya MR, et al. The function of the PduJ microcompartment shell protein is determined by the genomic position of its encoding gene. Mol microbiol. 2016;101(5):770–783. doi: 10.1111/mmi.13423 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0071] [71].Cheng S, Fan C, Sinha S, et al. The PduQ enzyme is an alcohol dehydrogenase used to recycle NAD+ internally within the Pdu microcompartment of Salmonella enterica. PLOS ONE. 2012;7(10):e47144. doi: 10.1371/journal.pone.0047144 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0072] [72].Penrod JT, Roth JR. Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica. J Bacteriol. 2006;188(8):2865–2874. doi: 10.1128/jb.188.8.2865-2874.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0073] [73].Fattinger SA, Sellin ME, Hardt WD. Salmonella effector driven invasion of the gut epithelium: breaking in and setting the house on fire. Curr Opin microbiol. 2021;64:9–18. doi: 10.1016/j.mib.2021.08.007 [DOI] [PubMed] [Google Scholar]

[cit0074] [74].Thiennimitr P, Winter SE, Winter MG, et al. Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota. Proc Natl Acad Sci USA. 2011;108(42):17480–17485. doi: 10.1073/pnas.1107857108 [DOI] [PMC free article] [PubMed] [Google Scholar]

[cit0075] [75].Rolfes RJ. Regulation of purine nucleotide biosynthesis: in yeast and beyond. Biochem Soc Trans. 2006;34(Pt 5):786–790. doi: 10.1042/bst0340786 [DOI] [PubMed] [Google Scholar]

PERMALINK

The putative AraC-type transcriptional regulator STM1082 facilitates Salmonella pathogenicity through modulation of intestinal invasion and intracellular replication

Shuai Ma

Wanwu Li

Xinyue Wang

Yuyang Sun

Houliang Guo

Jiaqi Ma

Lingyan Jiang

Roles

ABSTRACT

Introduction

Materials and methods

Ethics statement

Cell culture

Bacterial strains, plasmids, and growth conditions

Growth curve

Salmonella invasion of HeLa epithelial cells

Salmonella infection of macrophages

Bioluminescent reporter assays

Immunofluorescence staining

RNA isolation

Quantitative real-time PCR (qRT-PCR)

RNA sequencing and analyses

Mouse infection

Statistical analysis

Results

The expression of the STM1082 gene is up-regulated under conditions related to the intestinal environment and within macrophages

Figure 1.

STM1082 is crucial for Salmonella invasion into epithelial cells and replication within macrophages

Figure 2.

STM1082 is essential for the full virulence of Salmonella in mice

Figure 3.

Figure 4.

STM1082 up-regulates the expression of genes involved in 1, 2-propanediol and ethanolamine metabolism under intestinal-mimicking conditions

Table 1.

Figure 5.

STM1082 up-regulates the expression of genes involved in purine biosynthesis under macrophage-mimicking conditions

Table 2.

Figure 6.

Discussion

Supplementary Material

Acknowledgements

Funding Statement

Disclosure statement

Data availability statement

Supplemental data

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases