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Newly identified c-di-GMP pathway putative EAL domain gene STM0343 regulates stress resistance and virulence in Salmonella enterica serovar Typhimurium

Veterinary Research volume 56, Article number: 13 (2025) Cite this article

Abstract

S. Typhimurium is a significant zoonotic pathogen, and its survival and transmission rely on stress resistance and virulence factors. Therefore, identifying key regulatory elements is crucial for preventing and controlling S. Typhimurium. We performed transcriptomic analysis and screened for a c-di-GMP pathway key gene STM0343, a putative EAL domain protein with an unknown function. Our findings revealed that the deletion of this gene (269ΔSTM0343) led to a 29.85% increase in c-di-GMP. In terms of stress resistance, the strain 269ΔSTM0343 showed significant improvements compared to the wild strain WT269. Specifically, it exhibited increases of 95.74% in extracellular protein and 35.96% in exopolysaccharide production by upregulating the expression of relevant genes. As a result, the biofilm formation ability of 269ΔSTM0343 was enhanced by 21.54%, accompanied by a more pronounced red, dry, and rough colony morphology. 269ΔSTM0343 also showed a 19.03% decrease in motility due to the downregulation of flhD expression. As a result, 269ΔSTM0343 increased resistance to various antibiotics, as well as to acidic conditions, oxidative stress, and disinfectants. In terms of virulence, compared to WT269, the adhesion and invasive ability of 269ΔSTM0343 to HeLa cells was enhanced by onefold and 25.67%, respectively. In in vivo experiments, mice challenged with 269ΔSTM0343 experienced greater weight loss, and the bacterial loads in the spleen, liver, and intestines were elevated by fivefold, 30-fold, and 21-fold, respectively, accompanied by more severe pathological damage. Mechanistic studies revealed that the adhesion and invasion capacities of 269ΔSTM0343ΔCsgB decreased by 29.41% and 68.58%, respectively, compared to 269ΔSTM0343. Additionally, LacZ gene reporting indicated that STM0343 inhibited the expression of CsgB. This suggests that STM0343 suppresses virulence by downregulating CsgB expression. This study provides insights into the regulatory mechanisms by which STM0343 reduces the stress resistance and pathogenicity of S. Typhimurium.

Introduction

Salmonella is a significant zoonotic pathogen that poses serious risks to both animal and human health [1]. S. Typhimurium is one of the most important serotypes within the Salmonella genus, known for its high pathogenicity. It typically causes self-limiting illnesses such as vomiting, diarrhoea, and abdominal pain; however, severe cases can result in bacteremia and even death. Globally, S. Typhimurium is responsible for an estimated 21.7 million illnesses and 217 000 deaths [2].

Additionally, S. Typhimurium exhibits strong resistance to stress, including developing resistance to antibiotics and fungicides [3]. It also adapts to the host’s internal environment to facilitate growth and proliferation [3]. As a result, S. Typhimurium has become widespread and is the dominant serotype in many countries and regions, including China [4], Australia [5], and Africa [6]. Its strong adaptability to stress, high morbidity rates, and the challenges associated with prevention and control have established S. Typhimurium as a significant zoonotic pathogen concerning public health.

C-di-GMP is a second messenger that regulates various cellular processes in bacteria, contributing to stress adaptation and virulence [7]. It plays a crucial role in biofilm formation and motility in pathogens such as Escherichia coli [8], Vibrio vulnificus [9], and P. aeruginosa [10]. The synthesis and degradation of c-di-GMP are controlled by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which have GGDEF and EAL or HD-GYP domains, respectively [11, 12].

Different genes within this pathway have distinct regulatory functions. For example, AdrA, which contains a GGDEF domain, can promote biofilm formation by regulating the synthesis of exopolysaccharides [13], while STM1344, with an EAL domain, can inhibit the motility of S. Typhimurium [14]. The EAL-like protein STM1697 plays a role in regulating virulence [15]. In S. Typhimurium, a total of 22 genes associated with the c-di-GMP pathway have been identified [16].

Therefore, it is essential to explore new c-di-GMP pathway genes and understand their functions to gain insights into the gene regulatory network of S. Typhimurium. Our laboratory previously identified 20 differentially expressed genes in the c-di-GMP pathway by analysing strains with varying abilities to form biofilms [17]. Among these genes, STM0343, a hypothetical EAL domain gene, has an unclear role in biofilm formation, stress adaptation, and virulence in S. Typhimurium. Investigating STM0343 will enhance our understanding of the c-di-GMP regulatory network and identify potential control targets in S. Typhimurium.

Based on this, this study examined the role of the hypothetical EAL domain gene STM0343 in regulating c-di-GMP levels. Through phenotypic characterisation and mechanistic investigations, the study revealed how STM0343 affects biofilm formation, stress resistance, and virulence in S. Typhimurium. These findings lay the groundwork for screening potential targets and developing strategies for the prevention and control of S. Typhimurium.

Materials and methods

Strains, primers, and plasmids

The Salmonella strains utilised in this study included wild-type S. Typhimurium WT269, which was isolated from the joint fluid of patients at a hospital in Shanghai. HeLa cells were employed to evaluate the strains’ adhesion and invasive capabilities. Competent E. coli DH5α (Takara, China) was used for cloning experiments. All primers and plasmids used in this study are listed in Additional files 1, 2, and 3.

Construction of gene deletion mutant strains (269ΔSTM0343/269ΔCsgB/269ΔCsgBΔSTM0343) and STM0343 complemented transformant (269ΔSTM0343R)

The λRed-mediated mutagenesis method [18] used to generate the 269ΔSTM0343/269ΔCsgB/269ΔCsgBΔSTM0343 mutant strains. In this process, the kanamycin resistance gene, which is flanked by FRT sites, along with the upstream and downstream genes of STM0343/CsgB, was amplified using the primers STM0343-L1-F/R and CsgB-L2-F/R, respectively. The plasmid pKD4 served as the template for this amplification.

These amplified fragments were then transferred into WT269 competent cells, which had been induced with L-arabinose at a final concentration of 100 mmol/L. This induction allowed for the expression of pKD46, thereby facilitating recombination with the bacterial chromosomes. Plasmid pCP20 was subsequently transformed into homologous recombinant cells to remove the kanamycin cassette. The successfully mutated strains were confirmed using the primers STM0343-JD-F/R and CsgB-JD-F/R and were cultured at 42 °C to eliminate pCP20. The strain 269ΔCsgBΔSTM0343 was constructed by deleting the CsgB gene, using 269ΔSTM0343 as the genetic background. To create the STM0343 complemented transformant (269ΔSTM0343R), the STM0343 expression vector was constructed with pBAD-HisA and then electrotransferred into 269ΔSTM0343.

Determination of growth curves

The recovered WT269, 269ΔSTM0343, and 269ΔSTM0343R single colonies were picked and inoculated into LB broth, respectively. They were incubated overnight at 37 °C, with shaking at 180 rpm. The following day, the overnight culture was diluted into fresh LB broth, and the optical density at 600 nm (OD600nm) was adjusted to 0.1. The culture was continuously incubated at 37 °C with shaking at 180 rpm for 20 h. OD600nm values were measured at one-hour intervals, and the growth curves were plotted based on these measurements [19]. This experiment included three biological replicates.

Determination of bacterial c-di-GMP content

The extraction of c-di-GMP was performed using the method described by Spangler et al. [20], with minor modifications. First, the overnight cultures of WT269, 269ΔSTM0343, and 269ΔSTM0343R were diluted 1:100 into fresh LB broth and incubated for 8 h at 37 °C, with shaking at 180 rpm. The bacterial solution was then centrifuged, and the supernatant was discarded. The precipitate was washed three times with PBS, after which the bacteria were resuspended with PBS, adjusting the OD600nm to a consistent value. The organisms were broken down using a crushing method at 200 W for 10 s, followed by a 10 s pause, for a total processing time of 15 min. Afterwards, the mixture was centrifuged at 13 000 rpm for 2 min. The supernatant was discarded, and the remaining precipitate was washed with PBS. The precipitate was then resuspended in 2 mL of PBS and subjected to a water bath at 100 °C for 5 min. Following this, ice-cold ethanol (final concentration of 65%) was quickly added and extracted for 15 s. The mixture was then centrifuged at 13 000 rpm for 2 min, and the supernatant was collected to obtain the c-di-GMP sample. The c-di-GMP content in the sample was then measured using an ELISA kit from Shanghai Hengyuan Biological Technology Co., Ltd., and the results were quantified based on a standard curve. This experiment was conducted with three biological replicates.

Crystal violet staining to assess biofilm formation ability

Crystalline violet staining assay [21] was used to quantify the biofilm formation ability of WT269, 269ΔSTM0343, and 269ΔSTM0343R. The overnight cultured strains were diluted 1:100 in fresh unsalted LB broth. The diluted cultures were then inoculated into 96-well plates and incubated for 48 h at 30 °C. After discarding the planktonic bacteria, the cells in the wells were washed and fixed. A crystal violet solution was added to each well and allowed to stain for 15 min. The unbound dye was gently removed, and 200 µL of 33% acetic acid was added to each well to dissolve the crystal violet. Finally, the biofilm biomass was estimated by measuring the optical density at 595 nm (OD595 nm) using a microplate reader. This experiment was conducted with three biological replicates.

Observation of colony morphology

According to the previously reported method [22], 10 μL of the overnight cultured strains were inoculated onto pre-prepared salt-free LB agar plates containing 40 mg/mL of Congo red and 20 mg/mL of Coomassie Brilliant Blue G. The agar plates were then incubated at 27 °C for 5 days. At the end of the incubation period, the colours and roughness of the colonies on the agar plates were observed. This experiment was conducted with three biological replicates.

Quantification of the extracellular matrix

To determine the content of the biofilm-associated extracellular matrix, we followed method described by Dressaire et al. [23].

Colonies cultured on LB agar plates for 18 h were resuspended in 0.9% NaCl solution. After centrifugation, we collected the supernatant and mixed with it 99% cold ethanol. The mixture was then centrifuged at maximum speed for 20 min at 4 °C. The resulting pellet was dried and resuspended with ddH2O. Finally, we measured the concentrations of proteins and DNA in the samples using a NanoDrop 1000 (NanoDrop Technologies). To quantify the exopolysaccharide, 100 μL of the sample was taken and diluted to 1 mL with sterile deionised water. Next, a phenol solution was added, followed by concentrated sulfuric acid, with thorough mixing after each addition. The sample was allowed to stand for 10 min and then incubated in a water bath at 65 °C water bath for 18 min. After incubation, it was quickly cooled by placing it in ice until it reached room temperature. Finally, the absorbance at 490nm (OD490nm) was measured using a microplate reader, and the concentrations were determined based on a glucose calibration curve. This experiment was conducted with three biological replicates.

Motility assays

The motility of the strains WT269, 269ΔSTM0343, and 269ΔSTM0343R was assessed using the 0.3% agar plate method [21]. A volume of 10 μL from overnight cultures was inoculated in the centre of 0.3% semi-solid LB agar plates. The plates were then incubated at 37 °C, and the motility diameter of the strains in the agar was determined after 6 h. This experiment was conducted with three biological replicates.

RNA extraction and qRT-PCR (quantitative real-time reverse transcription PCR)

Overnight cultured WT269, 269ΔSTM0343, and 269ΔSTM0343R were diluted into fresh LB medium at a 1:100 ratio. The cultures were incubated at 37 °C with agitation at 180 rpm for 4 h. Following incubation, total bacterial RNA was then extracted using the RNA Extraction Kit (Omega Bio-tek, Norcross, GA, USA). The extracted RNA was then reverse transcribed into cDNA using the HiScript® III RT SuperMix for qPCR (+ gDNA WIper) according to the provided instructions. Finally, the expression levels of the genes BcsA, BcsB, CsgA, CsgB, which are involved in biofilm formation, as well as the genes fliA, flhC, flhD, which are related to bacterial motility, were analysed. This was done using a ChamQ Universal SYBR qPCR Master Mix kit (Novozan Biotechnology Co., Ltd., Nanjing, China) through qRT-PCR with the primers listed in Additional file 2. The 16S rRNA genes were used as an internal control for normalisation. This experiment was conducted with three biological replicates.

Determination of antibiotic susceptibility by broth microdilution method

Minimum Inhibitory Concentrations (MICs) of WT269, 269ΔSTM0343, and 269ΔSTM0343R against 15 antibiotics were determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). For the MIC assay, single colonies were transferred into MH broth and incubated at 37 °C, shaking at 180 rpm for 4 h until the OD600nm reached 0.5. A 1:100 dilution of the bacterial culture was then prepared in fresh MH broth. In a 96-well plate, 20 μL of antibiotics at an initial concentration of 10,240 μg/mL was added to 180 μL of MH broth. The antibiotics were then sequentially diluted in a twofold manner. Finally, 100 μL of the diluted bacterial solution was added to the various concentrations of antibiotics. The MIC values were measured after 16–18 h of static incubation at 37 °C. This experiment was conducted with three biological replicates.

Acid and oxidative stress assay

Overnight cultures of WT269, 269ΔSTM0343, and 269ΔSTM0343R were centrifuged at 6000 rpm for 5 min. The supernatant was discarded, and the organisms were washed three times with sterile PBS. After washing, the cells were resuspended in LB supplemented with acid (final pH = 4.4), oxidative (5 mM H2O2), and SDS disinfectant (0.25%). The OD600nm of the bacterial suspension was adjusted to 0.1. The cultures were then incubated at 37 °C with agitation at 220 rpm. OD600nm values were measured at 1-h intervals and growth curves were plotted [19]. This experiment was conducted with three biological replicates.

Cell adhesion and invasion assay

Overnight cultured strains were diluted 1:100 into fresh LB broth and incubated at 37 °C, with agitation at 220 rpm until the OD600nm was 0.6–0.8. After reaching the desired density, the bacterial culture was washed three times with PBS. The bacteria were then resuspended in DMEM. To assess cell adhesion, the resuspended bacteria were co-cultured with HeLa cells for 2 h at a multiplicity of infection (MOI) of 1:100. Subsequently, the cells were washed thrice with PBS to remove non-adherent bacteria. They were then treated with 0.1% Triton X-100 for 15 min to lyse the cells. The resulting cell lysates were serially tenfold multiplicative diluted with PBS, and colonies from the diluted lysates were counted using the plate decantation method. After co-culturing the bacteria with the cells for the cell invasion assay, the cells were washed three times with PBS containing 100 μg/mL gentamicin. Following the washes, DMEM containing 100 μg/mL of gentamicin was added, and the mixture was incubated for 1 h to eliminate any extracellular bacteria. Finally, the colonies were counted using the same method as described above. This experiment was conducted with three biological replicates.

In vivo infection of C57BL/6 mice

Animal experiments were conducted following the previously described protocols [1]. In brief, four-week-old SPF-grade C57BL/6 mice, purchased from the Guangdong Medical Laboratory Animal Center, Guangzhou, China, were divided into four groups of seven animals each. The mice were fasted for 24 h and water-deprived for 12 h before the challenge. They were then administered 1 × 108 CFU (100 μL) of WT269, 269ΔSTM0343, 269ΔSTM0343R by gavage, respectively. The last group of mice were each gavaged with 100 μL of PBS as a control.

The body weight of each mouse was recorded continuously for 7 days after the challenge, and on the seventh day, the mice were euthanised. Their intestines, livers, and spleens were then collected for bacterial colony counting. The bacterial load was calculated as bacterial CFU in 1 g of tissue (CFU/g). Additionally, pathological sections of the intestine, liver, and spleen were prepared for analysis. Based on previously reported methods, we conducted quantitative pathological scoring of the tissue sections. The scoring criteria were as follows: grade 0 for no damage, grade 1 for < 25% damage, grade 2 for 25–49% damage, grade 3 for 50–75% damage, grade 4 for > 75% damage [24]. Additionally, each mouse's body weight measurements and bacterial load counts were carried out in triplicate.

LacZ reporter gene fusion assay

In the previous report [25], the promoter of CsgB was amplified using the primers CsgB-pro-F/R. A LacZ gene reporter system targeting CsgB was constructed using pRCL as a vector. This constructed vector was then electro-transferred into WT269, 269ΔSTM0343, and 269ΔSTM0343R. The strains were incubated at 37 °C with shaking at 180 rpm for 8h. After adjusting the OD600nm to a consistent level, the cultured strains were disrupted using sonication. The OD600nm of the resulting liquid was measured, followed by centrifugation at 4 °C and 15 000 × g for 20 min. The supernatant was then collected. Finally, the activity of β-galactosidase in the supernatant was assessed using a kit from BOXBIO (Beijing, China). This experiment was conducted with three biological replicates.

Statistical analysis

All experiments in this study were performed in triplicate, and the data were analysed and visualised using GraphPad Prism V8.0 (GraphPad Inc., La Jolla, CA, USA). Multiple t-tests were performed to assess differences in the data. In the figures, asterisks denote statistical significance as follows: “*” indicates P ≤ 0.05, “* *” indicates P ≤ 0.01, and “* * *” indicates P ≤ 0.001.

Results

STM0343 decreased the level of c-di-GMP

The study conducted a growth curve analysis and found that STM0343 did not significantly affect the growth of S. Typhimurium (Figure 1A). Since STM0343 is a putative EAL domain protein, the present study investigated its effect on c-di-GMP levels. By measuring c-di-GMP levels in the wild strain WT269, as well as in the constructed deletion strain 269ΔSTM0343 and the complementation strain 269ΔSTM0343R (Additional file 4), it was discovered that the c-di-GMP content in the STM0343 deletion strain 269ΔSTM0343 was significantly increased by 29.85% (P < 0.01) compared to WT269 (Figures 1B and C). This suggests that STM0343 likely functions as a phosphodiesterase, degrading intracellular c-di-GMP.

Figure 1
figure 1

Detection of c-di-GMP content in bacteria. A Growth curves of WT269, 269∆STM0343, 269∆STM0343R. Bacteria with an initial OD600nm of 0.1 were cultured in LB broth, and OD600nm was measured hourly for a duration of 20 h. B A standard curve depicting c-di-GMP concentration in relation to OD450nm value, using c-di-GMP standards and the method outlined in the ELISA kit. The standard curve equation derived from this data is y = 0.016x + 0.0523 (R2 = 0.9887). C The c-di-GMP content of WT269, 269ΔSTM0343, 269ΔSTM0343R was detected according to the established standard curve, **P < 0.01.

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STM0343 reduces biofilm formation ability by inhibiting the production of extracellular proteins and exopolysaccharide

In this study, we investigated the effect of STM0343 on biofilm formation ability using crystal violet staining. It was found that the biofilm formation ability of the 269ΔSTM0343 strain was enhanced by 21.54% compared to the wild strain (P < 0.01, Figure 2A). This finding was further supported by the observation that the deletion of STM0343 led to a rougher colony morphology on Coomassie Brilliant Blue and Congo Red plates compared to the wild strain. The 269ΔSTM0343 strain exhibited a rough texture that spread across the surface of the moss and extended to its edges, resulting in wrinkled and irregular edge formations with increased magnitude. In contrast, the wild-type strain and the complemented strain (269ΔSTM0343R) exhibited smooth characteristics, unlike the roughness observed in the other samples (Figure 2B). These findings indicate that STM0343 plays a role in inhibiting biofilm formation.

Figure 2
figure 2

STM0343 reduces the biofilm formation ability of S. Typhimurium. A Detection of biofilm formation ability of the strains by crystal violet staining method; B Colony morphology was observed by incubation on Congo red (40 mg/mL) and Coomassie Brilliant Blue G (40 mg/mL) plates; C, D and E are the identification of extracellular protein, extracellular DNA and exopolysaccharide content of the strains, respectively; F Standard curve for the determination of exopolysaccharide content, **P < 0. 01, ***P < 0.001.

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To clarify the mechanism, we analysed the content of biofilm-associated extracellular matrix in WT269, 269ΔSTM0343, and 269ΔSTM0343R. Compared to the wild strain, the extracellular protein content in the strain 269ΔSTM0343 was significantly higher, showing an increase of 95.74% (P < 0.0001, Figure 2C). Additionally, exopolysaccharide content was determined using the constructed standard curve equation y = 0.913x + 0.0352 (R2 = 0.9941). It was found that the exopolysaccharide content in strain 269ΔSTM0343 was elevated by 35.96% compared to WT269 (Figures 2E and F). However, there were no significant differences in the extracellular DNA components between 269ΔSTM0343 and WT269 (P > 0.05, Figure 2D). At the gene level, the expression levels of extracellular protein-coding genes (CsgA and CsgB) and genes related to exopolysaccharide production (BcsA and BcsB) were significantly higher in 269ΔSTM0343, as analysed by QRT-PCR (Additional file 5). These findings suggest that STM0343 primarily reduces biofilm formation by inhibiting the production of extracellular proteins and exopolysaccharides.

STM0343 enhanced motility by promoting the expression of flagellar genes

To investigate the effect of STM0343 on bacterial motility, we measured the motility diameter of bacteria on 0.3% LB agar plates. The results showed that the motility diameter of 269∆STM0343 was reduced by 19.03% (P < 0.01) at the sixth hour compared to the wild strain WT269 (Figure 3). Additionally, at the gene level, the expression of the flagellar synthesis gene flhD (Additional file 5), which is associated with motility, was significantly reduced in the absence of STM0343 compared to the wild strain. This indicates that the deletion of STM0343 impairs the motility of S. Typhimurium by inhibiting flagellar synthesis.

Figure 3
figure 3

The effect of STM0343 on the motility of S. Typhimurium. A Motility distribution of WT269, 269ΔSTM0343 and 269ΔSTM0343R on 0.3% agar plates; B Measurement of motility diameter of WT269, 269ΔSTM0343 and 269ΔSTM0343R, **P < 0.01.

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Expression of STM0343 reduces the stress tolerance response of S. Typhimurium

Given that STM0343 is a putative c-di-GMP pathway gene and considering its regulatory role in biofilm formation, this study further investigates its function in S. Typhimurium’s response to various bactericidal environments.

To begin with, an antibiotic susceptibility test was conducted on 269ΔSTM0343, WT269, and 269ΔSTM0343R, involving 15 different antibiotics. The results revealed that 269ΔSTM0343 exhibited a sevenfold reduction in susceptibility to ceftazidime and ceftiofur, along with a onefold reduction in susceptibility to cefepime, cefotaxime, tetracycline, doxycycline, kanamycin, streptomycin, and nalidixic acid, when compared to the wild-type strain WT269 (Table 1).

Table 1 Effect of STM0343 on antibiotic susceptibility
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During stress tests involving acid, oxidative, and disinfectants, the growth of the strain was not inhibited after the deletion of STM0343; in fact, it was significantly faster. Additionally, the mutant strain exhibited higher OD600nm values than the wild-type strain during acid stress starting from the 8th hour, oxidative stress from the 7th hour, and SDS stress from the very first hour. This trend persisted until the 24th hour.

These findings suggest that STM0343 reduces the stress resistance of S. Typhimurium (Figure 4).

Figure 4
figure 4

Role of STM0343 in the stress response of S. Typhimurium. A–C are the effects of STM0343 on acid tolerance, oxygen tolerance, and SDS disinfectant tolerance of S. Typhimurium, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

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Deletion of STM0343 elevates the ability of S. Typhimurium to invade and adhere to HeLa cells in vitro

This study evaluated the virulence of S. Typhimurium by conducting cell adhesion and invasion assays using HeLa cells as an in vitro experimental model. The results indicated that the mutant strain 269ΔSTM0343 exhibited a onefold increase in adhesion ability and a 25.67% increase in invasion ability compared to the wild-type strain WT269 (P < 0.05). Furthermore, the complemented strain 269ΔSTM0343R restored both adhesion and invasion abilities to levels comparable to the wild strain (Figure 5). These findings tentatively suggest that STM0343 may play a role in inhibiting the virulence of S. Typhimurium.

Figure 5
figure 5

Effect of STM0343 on the cell invasion and adhesion abilities of S. Typhimurium. A Characterisation of STM0343 on the ability of the strain to adhesion to HeLa cells; B Characterisation of STM0343 on the ability of the strain to invade HeLa cells. *P < 0.05, **P < 0.01.

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Deletion of STM0343 enhances the virulence of S. Typhimurium in a mouse model of infection

To further investigate the role of STM0343 in the pathogenesis of S. Typhimurium, this study conducted in vivo infection experiments using mice. First, the body weight of the mice was monitored for seven consecutive days following the challenge. The results indicated that the body weight of mice in the blank control group (PBS) exhibited a steady increase. In contrast, the body weight of mice in the WT269, 269ΔSTM0343, and 269ΔSTM0343R challenged groups was lower than that of the blank control group. The body weight of mice challenged with WT269 and 269ΔSTM0343R increased initially over the first three days and then gradually stabilised. In contrast, the body weight of mice challenged with 269ΔSTM0343 began to decrease on day four and was significantly lower than that of the other two groups from day six onward (P < 0.05, Figure 6A).

Figure 6
figure 6

Infection of mice with S. Typhimurium strains in vivo. A Changes in body weight of mice stimulated by WT269, 269ΔSTM0343 and 269ΔSTM0343R; B–D Bacterial load in the spleen, liver and intestine of experimental mice challenged by WT269, 269ΔSTM0343 and 269ΔSTM0343R; E Histopathological section observations of livers, spleens and intestines of mice infected with WT269, 269ΔSTM0343 and 269ΔSTM0343R, the black arrows represent lesions. *P < 0.05, **P < 0.01, ***P < 0.001.

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The study also investigated the bacterial load in the spleen, liver, and intestine of the mice across the different groups. Compared to the WT269 challenge mice, the bacterial load in the intestine, spleen, and liver of mice infected with the 269ΔSTM0343 strain increased 21-fold, fivefold, and 30-fold, respectively (P < 0.01). However, there was no significant difference in bacterial load between the mice infected with the 269ΔSTM0343R strain and those infected with the wild-type strain (P > 0.05, Figures 6B–D).

Additionally, the pathological examination of the internal organs revealed that the mice in the challenge groups experienced varying degrees of pathological changes in the liver, spleen, and intestine compared to the blank control group. Notably, the internal organs of mice challenged with the 269ΔSTM0343 strain showed more severe damage than those challenged with the WT269 and 269ΔSTM0343R strains (Additional file 6).

The livers of the mice challenged with 269ΔSTM0343 displayed coagulation necrosis similar to that of the mice challenged with WT269, but they also exhibited significant inflammatory foci. In addition to the amyloid deposition observed in the spleen of the WT269 challenge mice, those challenged with 269ΔSTM0343 showed sparse lymphocytes and exposed endothelial cells in their spleens.

Examination of intestinal tissues revealed that all mice in the three challenge groups demonstrated necrosis and sloughing of intestinal epithelial cells. The intestinal damage in mice infected with the 269ΔSTM0343 strain was more severe compared to those challenged with the WT269 and 269ΔSTM0343R strains. Mice exposed to the 269ΔSTM0343 strain exhibited denatured, necrotic, and detached intestinal mucosal epithelial cells, fragmented tissue, and loss of histological morphology (Figure 6E). These findings suggest that STM0343 has a negative regulatory effect on the virulence of S. Typhimurium.

STM0343 affects the virulence of S. Typhimurium by regulating the expression of CsgB

Based on the above findings, STM0343 influences the expression of CsgB, which encodes curli fimbriae, a key virulence factor. To explore the relationship between STM0343 and CsgB in regulating the virulence of S. Typhimurium, this study conducted adhesion and invasion assays on HeLa cells. The 269ΔSTM0343 mutant strain displayed significantly improved cell invasion and adhesion capabilities compared to the wild-type strain WT269. However, when CsgB was simultaneously deleted in the 269ΔSTM0343 background (i.e., 269ΔSTM0343ΔCsgB, Additional file 7), both adhesion and invasion abilities decreased by 29.41% and 68.58%, respectively, compared to the 269ΔSTM0343 strain. These abilities returned to levels comparable to the wild-type strain (P > 0.05, Figures 7A and B), suggesting that STM0343 regulates the virulence of S. Typhimurium through CsgB.

Figure 7
figure 7

STM0343 affects S. Typhimurium virulence by regulating CsgB. A, B Cell adhesion and invasion assay. C LacZ reporter system to detect regulation of CsgB expression by STM0343. *P < 0.05, **P < 0.01, ***P < 0.001.

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To further investigate whether STM0343 has a regulatory effect on CsgB expression, we performed a LacZ transcriptional reporter assay (Additional file 8). We measured β-galactosidase activity as an indicator of CsgB activation. As expected, β-galactosidase activity was elevated by 15.59% in the 269ΔSTM0343 strain compared to the wild-type strain WT269 (P < 0.001). Additionally, in the complemented strain 269ΔSTM0343R, β-galactosidase activity was restored to the level observed in WT269 (Figure 7C). These results suggest that STM0343 inhibits the expression of CsgB, thereby reducing the virulence of S. Typhimurium.

Discussion

Stress adaptation and effective virulence are key factors in the widespread occurrence of S. Typhimurium, presenting significant challenges for its prevention and control. A crucial regulatory molecule involved in these processes is c-di-GMP, which is commonly found in bacteria and regulates various life processes, including biofilm formation and virulence. Therefore, studying the genes involved in the c-di-GMP pathway is essential for identifying potential targets for preventing and controlling S. Typhimurium.

Preliminary transcriptomics studies, which involved constructing strains with varying abilities to form biofilms, revealed that c-di-GMP is essential for the biofilm formation of S. Typhimurium. Additionally, the gene STM0343, a putative EAL domain protein, was identified as a key player in the c-di-GMP pathway [17].

In this study, we further investigated the function of STM0343. Our results interesting showed that STM0343 decreased c-di-GMP levels in bacteria, independent of bacterial growth. Previous studies have recognised STM0343 as a potential EAL domain gene [26], corroborating earlier findings that EAL proteins can reduce c-di-GMP levels [11]. This suggests that STM0343 may function as a phosphodiesterase. However, further experiments, such as point mutation analysis, are necessary to confirm this.

We further investigated the role that STM0343 plays in the biofilm formation ability of S. Typhimurium. The crystal violet assay demonstrated that the deletion of STM0343 enhanced biofilm formation. Additionally, the Congo red plate assay showed that the colony morphology of 269ΔSTM0343 appeared drier and rougher. This suggests an increased production of the extracellular matrix, which facilitates biofilm formation [27].

These results indicate that STM0343 functions as an inhibitor of biofilm formation by S. Typhimurium. Biofilm is primarily composed of bacterial communities that are encased in an extracellular matrix, which includes exopolysaccharides, extracellular proteins and extracellular DNA (eDNA) [28]. To better understand STM0343’s regulatory role in S. Typhimurium biofilm formation, we quantified the extracellular matrix associated with the biofilm. We found that STM0343 primarily inhibits the production of extracellular proteins and exopolysaccharides.

Extracellular proteins are crucial for stabilising the biofilm structure and for adhering bacteria to surfaces [28]. As a result, the levels of these extracellular proteins were elevated in the strain 269ΔSTM0343. Furthermore, the deletion of STM0343 caused a significant increase in the expression levels of the CsgA and CsgB genes, both of which are essential for encoding extracellular proteins [29, 30].

Exopolysaccharides are believed to play a crucial role in the adhesion of bacteria to biotic or abiotic surfaces during the formation of biofilms. In this study, we found that the exopolysaccharide content was significantly higher in the strain 269ΔSTM0343 compared to the wild type. Furthermore, cellulose, which is the main exopolysaccharide component of the biofilm matrix, along with its encoding genes BcsA and BcsB, showed increased levels of expression in the 269ΔSTM0343 strain.

The results indicated that STM0343 reduces the ability of S. Typhimurium to form biofilms primarily by limiting the synthesis of extracellular proteins and exopolysaccharides. Interestingly, previous studies have shown that deleting several EAL domain genes does not impact biofilm formation [31]. This emphasises the critical role of STM0343, identified in this study, in regulating c-di-GMP levels and biofilm formation. Since biofilms are an important adaptive mechanism for bacteria [32], our findings underscore the necessity for further investigation into the mechanisms behind STM0343’s regulatory effects.

The deletion of STM0343 was found to inhibit motility of S. Typhimurium, primarily due to a reduction in flagellar synthesis. This finding is supported by a significant decrease in flhD expression, a crucial regulator of flagellar synthesis, as noted in previous studies [33].

Interestingly, compensatory mechanisms may arise in response to the loss of STM0343. We observed an increase in the expression of fimbria-like adhesins (CsgA and CsgB). This finding is consistent with other research suggesting that when flagellar motility is compromised, bacteria often enhance their adhesive properties to maintain their ecological niches [34].

This shift highlights a strategic transition: motility allows bacteria to access optimal habitats, such as nutrient-rich environments [35], and facilitates their transition from a planktonic state to a stationary state, ultimately leading to biofilm formation [36]. Therefore, STM0343 is a novel molecule that inhibits the biofilm formation of S. Typhimurium.

Biofilm formation provides a protective effect for S. Typhimurium; however, our findings indicate that STM0343 inhibits biofilm formation. To further investigate the role of STM0343 in the resistance of S. Typhimurium to adverse environmental factors, we first examined its effect on antibiotic resistance. The results showed that the strain 269ΔSTM0343 exhibited a significantly increased resistance to multiple antibiotics, including ceftazidime, ceftiofur, cefepime, cefotaxime, tetracycline, doxycycline, kanamycin, streptomycin, and nalidixic acid. Cephalosporins and fluoroquinolones are commonly used to treat severe Salmonella infections [37].

Previous studies have shown that bacteria capable of forming biofilms often have increased tolerance to antibiotics, making treatment more challenging [32]. Therefore, inhibiting biofilm formation by STM0343 could be a critical factor in managing antibiotic resistance in S. Typhimurium.

In addition to antibiotic resistance, we found that the deletion of STM0343 increased resistance to SDS disinfectants and enhanced tolerance to oxidative and acidic stress. SDS disinfectants are commonly used to eliminate environmental bacteria, while oxidative and acid stress act as antibacterial mechanisms in mammalian cells, helping to rapidly eliminate bacterial pathogens [38, 39]. Therefore, STM0343 plays a crucial role in eliminating stress adaptation in S. Typhimurium.

Our study revealed that STM0343 plays a role in regulating the virulence of S. Typhimurium. Firstly, through in vitro cell invasion and adhesion assays, we found that the deletion of STM0343 significantly enhanced adherence to HeLa cells compared to the wild-type strain. This increased adherence ability allows the bacteria to persist in the extracellular space and facilitates their invasion into host cells [40]. Interestingly, the deletion of STM0343 enhanced the invasion ability of S. Typhimurium to HeLa cells, indicating that STM0343 may inhibit the virulence of S. Typhimurium.

Additionally, our in vivo experiments using a mouse model support this conclusion. Mice treated with 269ΔSTM0343 exhibited noticeable weight loss, and there was a significant increase in bacterial load in the intestines, liver, and spleen, along with greater tissue damage in these organs compared to the wild-type strain WT269. Effective virulence is essential for S. Typhimurium to disseminate widely, and the coordinated expression of genes in pathogenic bacteria is necessary to enhance the efficiency of virulence processes [19].

Our findings emphasise the significant role of STM0343 in regulating the virulence of S. Typhimurium.

Our study investigated the mechanism by which STM0343 regulates virulence. We found that STM0343 influences the expression of CsgB, a regulatory gene crucial for the synthesis of curli fimbriae and an important factor in virulence [41]. Previous studies have identified key factors that affect the pathogenicity of S. Typhimurium by regulating downstream virulence genes [42]. Based on this information, we hypothesised that STM0343 modulates the virulence of S. Typhimurium by regulating CsgB.

To test this hypothesis, we created a double deletion mutant of STM0343 and CsgB (269ΔSTM0343ΔCsgB). Our observations indicated that the enhanced pathogenicity of 269ΔSTM0343 was significantly reduced and returned to the levels seen in the wild-type strain after the simultaneous deletion of CsgB (269ΔSTM0343ΔCsgB).

To further confirm the regulatory effect of STM0343 on CsgB, we conducted a LacZ reporter gene fusion assay to evaluate CsgB activity by indirectly measuring β-galactosidase levels [43]. The results showed that the β-galactosidase content in the STM0343 knockout mutant was significantly higher than that in the wild-type WT269. Additionally, reintroducing the STM0343 gene restored β-galactosidase levels to those of the wild-type. This finding suggests that STM0343 can repress the expression of the CsgB.

These findings are consistent with the qRT-PCR results mentioned above and further support the conclusion that STM0343 reduces the pathogenicity of S. Typhimurium by inhibiting the expression of the curli fimbriae gene CsgB. In summary, this study has identified the key gene STM0343 that regulates the stress adaptability and virulence of S. Typhimurium. Future research should explore the molecular mechanisms that underlie the regulatory factors influencing the optimal biological functions of STM0343. This understanding will be valuable in addressing the challenges of controlling S. Typhimurium from the perspective of stress adaptability.

Stress adaptation and virulence are critical for the survival and proliferation of S. Typhimurium. In this study, we identified a novel EAL domain gene, STM0343, which reduces the levels of c-di-GMP while also affecting the stress resistance and virulence of S. Typhimurium. Regarding stress resistance, STM0343 enhances bacterial motility by promoting the expression of flagellar synthesis genes. Additionally, it suppresses the production of extracellular proteins by downregulating the expression of CsgB and CsgA, as well as exopolysaccharides, by decreasing the expression of BcsA and BcsB. This, in turn, inhibits biofilm formation. Ultimately, STM0343 decreases the resistance of S. Typhimurium to various antibiotics, acids, oxidative stress, and disinfectants. Additionally, STM0343 diminishes the virulence of S. Typhimurium by inhibiting the expression of the virulence factor CsgB. This study represents a significant advancement in the functional identification of c-di-GMP pathway genes and opens up new possibilities for developing prevention and control strategies against S. Typhimurium.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

References

  1. Zhang J, Peng Z, Chen K, Zhan Z, Shen H, Feng S, Gou H, Qu X, Ziemann M, Layton DS, Wang X, Chen H, Wu B, Xu X, Liao M (2023) Genomic Characterization of Salmonella enterica serovar Weltevreden associated with human diarrhea. Microbiol Spectr 11:e0354222

    Article  PubMed  Google Scholar 

  2. Qin Y, Hasman H, Aarestrup FM, Alwathnani HA, Rensing C (2014) Genome sequences of three highly copper-resistant Salmonella enterica subsp. I serovar Typhimurium strains isolated from pigs in Denmark. Genome Announc 2:e01334-14

    Article  PubMed  PubMed Central  Google Scholar 

  3. Winter SE, Thiennimitr P, Winter MG, Butler BP, Huseby DL, Crawford RW, Russell JM, Bevins CL, Adams LG, Tsolis RM, Roth JR, Bäumler AJ (2010) Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467:426–429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang Y, Liu Y, Lyu N, Li Z, Ma S, Cao D, Pan Y, Hu Y, Huang H, Gao GF, Xu X, Zhu B (2023) The temporal dynamics of antimicrobial-resistant Salmonella enterica and predominant serovars in China. Natl Sci Rev 10:nwac269

    Article  CAS  PubMed  Google Scholar 

  5. Williamson DA, Lane CR, Easton M, Valcanis M, Strachan J, Veitch MG, Kirk MD, Howden BP (2018) Increasing antimicrobial resistance in nontyphoidal Salmonella isolates in Australia from 1979 to 2015. Antimicrob Agents Chemother 62:e02012-17

    Article  PubMed  PubMed Central  Google Scholar 

  6. Reddy EA, Shaw AV, Crump JA (2010) Community-acquired bloodstream infections in Africa: a systematic review and meta-analysis. Lancet Infect Dis 10:417–432

    Article  PubMed  PubMed Central  Google Scholar 

  7. Jenal U, Reinders A, Lori C (2017) Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol 15:271–284

    Article  CAS  PubMed  Google Scholar 

  8. Méndez-Ortiz MM, Hyodo M, Hayakawa Y, Membrillo-Hernández J (2006) Genome-wide transcriptional profile of Escherichia coli in response to high levels of the second messenger 3’,5’-cyclic diguanylic acid. J Biol Chem 281:8090–8099

    Article  PubMed  Google Scholar 

  9. Nakhamchik A, Wilde C, Rowe-Magnus DA (2008) Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus. Appl Environ Microbiol 74:4199–4209

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ueda A, Wood TK (2009) Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5:e1000483

    Article  PubMed  PubMed Central  Google Scholar 

  11. Christen M, Christen B, Folcher M, Schauerte A, Jenal U (2005) Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J Biol Chem 280:30829–30837

    Article  CAS  PubMed  Google Scholar 

  12. Paul R, Weiser S, Amiot NC, Chan C, Schirmer T, Giese B, Jenal U (2004) Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 18:715–727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Römling U (2005) Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae. Cell Mol Life Sci 62:1234–1246

    Article  PubMed  PubMed Central  Google Scholar 

  14. Simm R, Remminghorst U, Ahmad I, Zakikhany K, Römling U (2009) A role for the EAL-like protein STM1344 in regulation of CsgD expression and motility in Salmonella enterica serovar Typhimurium. J Bacteriol 191:3928–3937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ahmad I, Wigren E, Le Guyon S, Vekkeli S, Blanka A, El Mouali Y, Anwar N, Chuah ML, Lünsdorf H, Frank R, Rhen M, Liang ZX, Lindqvist Y, Römling U (2013) The EAL-like protein STM1697 regulates virulence phenotypes, motility and biofilm formation in Salmonella typhimurium. Mol Microbiol 90:1216–1232

    Article  CAS  PubMed  Google Scholar 

  16. Ahmad I, Cimdins A, Beske T, Römling U (2017) Detailed analysis of c-di-GMP mediated regulation of csgD expression in Salmonella typhimurium. BMC Microbiol 17:27

    Article  PubMed  PubMed Central  Google Scholar 

  17. Chen K, Li L, Zhou Z, Wang N, Dai C, Sun D, Li J, Xu C, Liao M, Zhang J (2023) BolA promotes the generation of multicellular behavior in S. Typhimurium by regulating the c-di-GMP pathway genes yeaJ and yhjH. Int J Food Microbiol 411:110518

    Article  PubMed  Google Scholar 

  18. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mil-Homens D, Barahona S, Moreira RN, Silva IJ, Pinto SN, Fialho AM, Arraiano CM (2018) Stress response protein BolA influences fitness and promotes Salmonella enterica serovar Typhimurium virulence. Appl Environ Microbiol 84:e02850-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Spangler C, Böhm A, Jenal U, Seifert R, Kaever V (2010) A liquid chromatography-coupled tandem mass spectrometry method for quantitation of cyclic di-guanosine monophosphate. J Microbiol Methods 81:226–231

    Article  CAS  PubMed  Google Scholar 

  21. Gómez-Baltazar A, Vázquez-Garcidueñas MS, Larsen J, Kuk-Soberanis ME, Vázquez-Marrufo G (2019) Comparative stress response to food preservation conditions of ST19 and ST213 genotypes of Salmonella enterica serotype Typhimurium. Food Microbiol 82:303–315

    Article  PubMed  Google Scholar 

  22. Zhang Z, Du W, Wang M, Li Y, Su S, Wu T, Kang Y, Shan X, Shi Q, Zhu G (2020) Contribution of the colicin receptor CirA to biofilm formation, antibotic resistance, and pathogenicity of Salmonella Enteritidis. J Basic Microbiol 60:72–81

    Article  CAS  PubMed  Google Scholar 

  23. Dressaire C, Moreira RN, Barahona S, Alves de Matos AP, Arraiano CM (2015) BolA is a transcriptional switch that turns off motility and turns on biofilm development. MBio 6:e02352-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nesterenko LN, Kobets NV, Balunets DV (2012) Model of chronic salmonellosis: parameters of infection and immune response in inbred mice genetically variable in susceptibility to salmonellosis. Zh Mikrobiol Epidemiol Immunobiol 4:9–14 (in Russian)

    Google Scholar 

  25. Fu L, Xu J, Han X, Yang H, Lai Y, Huang Z, Gong J (2021) Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene. Sheng Wu Gong Cheng Xue Bao 37:321–330 (in Chinese)

    CAS  PubMed  Google Scholar 

  26. Ahmad I, Lamprokostopoulou A, Le Guyon S, Streck E, Barthel M, Peters V, Hardt WD, Römling U (2011) Complex c-di-GMP signaling networks mediate transition between virulence properties and biofilm formation in Salmonella enterica serovar Typhimurium. PLoS ONE 6:e28351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cimdins A, Simm R (2017) Semiquantitative analysis of the red, dry, and rough colony morphology of Salmonella enterica Serovar Typhimurium and Escherichia coli using Congo red. Methods Mol Biol 1657:225–241

    Article  CAS  PubMed  Google Scholar 

  28. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633

    Article  CAS  PubMed  Google Scholar 

  29. Hammer ND, Schmidt JC, Chapman MR (2007) The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization. Proc Natl Acad Sci USA 104:12494–12499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Barnhart MM, Chapman MR (2006) Curli biogenesis and function. Annu Rev Microbiol 60:131–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen Y, Lv M, Liao L, Gu Y, Liang Z, Shi Z, Liu S, Zhou J, Zhang L (2016) Genetic modulation of c-di-GMP turnover affects multiple virulence traits and bacterial virulence in rice pathogen Dickeya zeae. PLoS ONE 11:e0165979

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ciofu O, Tolkernielsen T (2010) Antibiotic tolerance and resistance in biofilms. In: Bjarnsholt T, Jensen P, Moser C, Høiby N (eds) Biofilm infections. Springer, New York, pp 215–229

    Chapter  Google Scholar 

  33. De Lay N, Gottesman S (2012) A complex network of small non-coding RNAs regulate motility in Escherichia coli. Mol Microbiol 86:524–538

    Article  PubMed  PubMed Central  Google Scholar 

  34. Prigent-Combaret C, Prensier G, Le Thi TT, Vidal O, Lejeune P, Dorel C (2000) Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid. Environ Microbiol 2:450–464

    Article  CAS  PubMed  Google Scholar 

  35. Fenchel T (2002) Microbial behavior in a heterogeneous world. Science 296:1068–1071

    Article  CAS  PubMed  Google Scholar 

  36. Karatan E, Watnick P (2009) Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Mol Biol Rev 73:310–347

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jiang Y, Wang ZY, Li QC, Lu MJ, Wu H, Mei CY, Shen PC, Jiao X, Wang J (2023) Characterization of extensively drug-resistant Salmonella enterica serovar Kentucky sequence type 198 isolates from chicken meat products in Xuancheng. China Microbiol Spectr 11:e0321922

    Article  PubMed  Google Scholar 

  38. Janssen R, Van Der Straaten T, Van Diepen A, Van Dissel JT (2003) Responses to reactive oxygen intermediates and virulence of Salmonella typhimurium. Microb Infect 5:527–534

    Article  CAS  Google Scholar 

  39. Ryan D, Ojha UK, Jaiswal S, Padhi C, Suar M (2016) The small RNA DsrA influences the acid tolerance response and virulence of Salmonella enterica Serovar Typhimurium. Front Microbiol 7:599

    Article  PubMed  PubMed Central  Google Scholar 

  40. Saldaña Z, Xicohtencatl-Cortes J, Avelino F, Phillips AD, Kaper JB, Puente JL, Girón JA (2009) Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli. Environ Microbiol 11:992–1006

    Article  PubMed  PubMed Central  Google Scholar 

  41. Sheng H, Xue Y, Zhao W, Hovde CJ, Minnich SA (2020) Escherichia coli O157:H7 curli fimbriae promotes biofilm formation, epithelial cell invasion, and persistence in cattle. Microorganisms 8:580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pati NB, Vishwakarma V, Jaiswal S, Periaswamy B, Hardt WD, Suar M (2013) Deletion of invH gene in Salmonella enterica serovar Typhimurium limits the secretion of Sip effector proteins. Microbes Infect 15:66–73

    Article  CAS  PubMed  Google Scholar 

  43. Aviv G, Gal-Mor O (2018) lacZ reporter system as a tool to study virulence gene regulation in bacterial pathogens. Methods Mol Biol 1734:39–45

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors sincerely appreciate Prof. Zhangyong Ning for his valuable guidance and assistance in the preparation of pathological slides.

Funding

This study was funded by the National Key Research and Development Program (2022YFD1800400); National Natural Science Foundation of China (82000011); National Natural Science Foundation of China (31972762); Specific university discipline construction project (Grant 2023B10564003); Walmart Foundation (61626817 & 52514289) and supported by Walmart Food Safety Collaboration Center; National Broiler Industry Technology System Project (CARS-41). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.

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  1. Kaifeng Chen and Lili Li contributed equally to this work.

Authors and Affiliations

  1. National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Key Laboratory of Zoonoses, Ministry of Agriculture, Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China

    Kaifeng Chen, Lili Li, Nanwei Wang, Zhouping Zhou, Peng Pan, Chenggang Xu, Dage Sun, Jiayi Li, Changzhi Dai, Ming Liao & Jianmin Zhang

  2. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China

    Ming Liao

  3. National Health Commission (NHC) Key Laboratory of Tropical Disease Control, School of Tropical Medicine, Hainan Medical University, Haikou, China

    Dai Kuang

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Contributions

KC and LL: designing and conducting experiments, writing original manuscripts, formal analysis; NW. W, ZZ. PP: visualization and methodology; CX: investigation; DS, JL and CD: Software and visualization; DK: funding acquisition; ML: supervision; JZ: project administration, funding acquisition and writing—review and editing. All authors read and approved the final manuscript.

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Correspondence to Jianmin Zhang.

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The mouse experimental protocols received approval by the Laboratory Animal Centre of South China Agricultural University and the Laboratory Animal Ethics Committee of South China Agricultural University (NO. 2024F245).The experimental animals were housed in the same environment and all procedures were conducted in compliance with the China Regulations for the Administration of Affairs Concerning Experimental Animals (1988) and Regulations for the Administration of Affairs Concerning Experimental Animals in Guangdong province (2010).

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Supplementary Information

Additional file 1: List of primers used for PCR.

Additional file 2

: List of primers used for qRT-PCR.

Additional file 3: List of plasmids used in this work.

Additional file 4: Construction of

STM0343 deletion mutant and deletion complementation strains. A Identification of the STM0343 deletion mutation, lane 1 is the successful mutant strain with a band size of 740bp, lane 2 is the wild strain with a band size of 2320bp. B and C Construction of the STM0343 complementation strain, B Construction of STM0343 expression vector using plasmid pBAD as a vector, lane 1 is an empty vector, and the band size is 420bp, lane 2 for the successful construction of the expression vector, with a band size of 2100 bp. C Validation of successful transfer of STM0343 expression vector into the strain. Lanes 1, 2, and 3 are all successfully constructed STM0343 complementation strains with a band size of 2100bp. M: DL5000 DNA Marker.

Additional file 5: qRT-PCR analysis of the effect of

STM0343 on gene expression related to biofilm formation and motility.

Additional file 6: Pathology scores in liver, spleen, and intestinal tissues of WT269, 269Δ

STM0343, and 269ΔSTM0343R-infected mice. *P < 0. 05, ***P < 0.001.

Additional file 7: Construction of

CsgB deletion strain, STM0343 and CsgB double deletion strain. A Identification of CsgB deletion strains, lanes 1–10 are successful deletion of CsgB strains. B Deletion of CsgB in the genetic background of 269ΔSTM0343, lanes 1–10 are successfully constructed CsgB and STM0343 double deletion mutants (269ΔCsgBΔSTM0343). + represents WT269 as a positive control, M: DL1000 DNA Marker.

Additional file 8: Construction of the LacZ gene reporter system.

A PCR amplification of the CsgB promoter. 1, 2, 3 are successfully amplified samples with band sizes of 885bp, M: DL1000 DNA Marker; B Purification of CsgB promoter after double enzyme digestion, M: DL5000 DNA Marker; C Plasmid PRCL purification after double digestion, M: DL5000 DNA Marker; D PCR validation of the recombinant plasmid PRCL-CsgB after transformation into WT269, 269ΔSTM0343, 269ΔSTM0343R, 1–4 represent the successful transfer of recombinant plasmid into the WT269; 5–8 represent the successful transfer of recombinant plasmid into the 269ΔSTM0343; numbers 9–14 represent the successful transfer of recombinant plasmid into the 269ΔSTM0343R. + , for plasmid PRCL amplification band, M: DL2000 DNA Marker.

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Chen, K., Li, L., Wang, N. et al. Newly identified c-di-GMP pathway putative EAL domain gene STM0343 regulates stress resistance and virulence in Salmonella enterica serovar Typhimurium. Vet Res 56, 13 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13567-024-01437-0

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Veterinary Research

ISSN: 1297-9716