Inhibition of the type III secretion system by syringaldehyde protects mice from Salmonella enterica serovar Typhimurium

Abstract The invasiveness of Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely associated with the Salmonella pathogenicity island (SPI)‐encoded type Ⅲ secretion system (T3SS), which can directly inject a series of effector proteins into eukaryotic cells to enable bacterial infection. In this study, syringaldehyde was identified as an effective inhibitor of the S. Typhimurium T3SS using an effector protein‐lactamase fusion reporter system. Syringaldehyde treatment could inhibit the expression of important effector proteins (SipA, SipB and SipC) at a concentration of 0.18 mM without affecting bacterial growth. Additionally, significant inhibition of bacterial invasion and cellular injury was observed following the syringaldehyde treatment in the co‐infection system of HeLa cells and S. Typhimurium. Furthermore, treatment with syringaldehyde provided systemic protection to mice infected with S. Typhimurium, reducing mortality (40.00%) and bacterial loads and relieving caecal damage and systemic inflammation. The results presented in this study indicate that syringaldehyde significantly affects T3SS activity and is a potential leading compound for treating S. Typhimurium infections.

successful infection caused by this bacterium requires the delivery of effector proteins into host cells via two type III secretion systems (T3SSs) encoded by the distinct Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and SPI-2). 5,6 Notably, the translocation of effector proteins via the SPI-1-associated T3SS is largely involved in facilitating bacterial invasion of host cells, 7 whereas SPI-2 T3SS-delivered effector proteins are primarily associated with promoting intracellular survival and replication. 8 In a search for bacterial effector proteins, a previous study observed that SipA was necessary for efficient bacterial entry into epithelial cells. 2 The mutant sipA was also impaired in its ability to be internalized by cultured epithelial cells after short infection times. 9 The transport of bacterial effector proteins across the host cell membrane is facilitated by a subset of Salmonella effector proteins known as translocators, including SipB, SipC and SipD. 10,11 These proteins form a channel in the host cell membrane that enables the passage of bacterial effector proteins into the host cell. 10 Studies have shown that the complex formed by SipC and SipB is essential for the insertion of both proteins into the host cell membrane during Salmonella invasion. 12 Several trials have shown that mutations in genes of the inv operon, such as invA and invG, result in a reduction of virulence and colonization of Salmonella by the oral route. 13,14 The complexity of this molecular machine makes fighting infections a challenge.
The high incidence of antibiotic resistance in this pathogen makes the use of traditional therapies inefficient. 15 Innovative therapeutic strategies aimed at inhibiting the T3SS of S. Typhimurium as well as alternatives to classical antibiotics are highly attractive, as they may reduce the severity of infection and improve antibacterial immune responses as well as representing attractive therapies for antibiotic-resistant bacteria with a similar virulence system. Thus, T3SS is increasingly being proposed and explored as an attractive drug target for developing novel antibacterial agents. 15, 16 We recently used a protein translocation assay and Western blotting to screen a series of natural plant compounds for their effect on the translocation and expression of T3SS effector proteins in vitro and identified a novel small terpenoid inhibitor, syringaldehyde, that effectively suppressed the activity of the T3SS in S. Typhimurium. In addition, we determined that syringaldehyde reduced the mortality and bacterial loads and relieved caecal damage and systemic inflammation in vivo.
The preliminary mechanism of action determined for syringaldehyde is also discussed. In summary, syringaldehyde shows strong potential as a leading compound to inhibit the T3SS and pathogenicity of S. Typhimurium.

| Bacterial strains and reagents
The S. Typhimurium strain SL1344 and the invA mutant used in this study were kindly provided by Profs. Zhaoqing Luo and Daoguo Zhou from the Immunology and Infectious Diseases laboratory and the Department of Biological Sciences at Purdue University respectively. In addition, we used a S. Typhimurium strain expressing a SipA-lactamase fusion and S. Typhimurium SipA-3×Flag and SipB-3×Flag strains that were constructed by our laboratory.
Unless otherwise specified, the S. Typhimurium strains were cultured at 37°C in Luria-Bertani (LB) medium containing 0.3 m NaCl. All of the candidate drugs (purity > 98%) were purchased from Chengdu Heibpurify Co., Ltd. (Chengdu, China) and were dissolved in dimethyl sulphoxide (DMSO, Sigma-Aldrich, United States).

| Effector protein expression assay
The expression of effector proteins was assayed by Western blotting. The S. Typhimurium strains (SL1344, SipA-3×flag and SipB-3×flag) were grown overnight in LB broth. Next, the cells were subcultured (1:100) into fresh LB and were grown for 4 hours with different concentrations of syringaldehyde. Subsequently, cells from specific volumes of the cultures (determined based on OD600 nm measurements) were collected by centrifugation at 13800 g for 5 minutes. The cell pellets were resuspended in 1 × sodium dodecyl sulphate (SDS)-loading buffer and boiled for 5 minutes, after which the samples were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Immunoblotting analyses were performed using the appropriate primary antibodies (an anti-Flag-tag mouse monoclonal antibody (  17 The blots were detected using the enhanced chemiluminescence (ECL) method and were scanned with a Tanon-4200 Image System (Tanon, China).

| Lactate dehydrogenase release assay
HeLa cells were plated into 96-well plate at a density of 2×10 4 cells/ well and were incubated at 37°C in a 5% CO 2 incubator for 16-18 hours before infection. After washing the cells three times with PBS, they were infected at an MOI of 100 with S. Typhimurium and cultured with different concentrations of syringaldehyde for 5 hours in DMEM. Subsequently, the cultures were centrifuged at 139.8 g for 10 min and the LDH release was then measured in culture supernatants on a microplate reader at 490 nm using an LDH Cytotoxicity Assay Kit according to the manufacturer's instructions (Roche, Switzerland). The percentage of LDH release was calculated as described in a previous study. 17 The LDH release from the HeLa cells incubated with syringaldehyde was measured at an effective concentration range (0.0225-0.18 mmol/L) in independent tests to evaluate the cytotoxicity of syringaldehyde.

| Adhesion, invasion, replication assay and immunofluorescence
The adhesion, invasion and replication of S. Typhimurium in Hela cells were verified using a gentamicin protection assay and was optimized according to the method described before. 18,19 HeLa cells were plated into 24-well plates at a density of 5×10 5 cells/well. Overnight bacterial cultures were diluted 20-fold into fresh LB with or without syringaldehyde at 37°C for 4 hours. After washing, HeLa cells were infected at a MOI of 100 and incubated at 37°C for 30 minutes. The HeLa cells were washed three times by PBS to clear away unadhered bacteria. Subsequently, the cells were permeabilized with 0.2% saponin for 10 minutes, 20,21 and the adhesive bacteria were counted by plating on LB agar plates. Following washing, save the plate labelled with 'invasion' for further treatment. DMEM containing 100 μg/ml gentamicin was added to each well of the plates in order to kill the extracellular Salmonella. The plates were incubated at 37°C, 5% CO 2 for an additional 1 hour. After washing three times, the cells were permeabilized with 0.2% saponin for 10 minutes and the invasive bacteria were counted by plating on LB agar plates. Following washing, save the plate labelled with 'replication' for further treatment.
DMEM containing 10 μg/ml gentamicin was added to the 'replication' plate to maintain clearance of extracellular Salmonella in the medium. The plate was incubated at 37°C, 5% CO 2 for an additional 22.5 hours. After washing three times, the cells were permeabilized with 0.2% saponin for 10 minutes and the invasive bacteria were counted by plating on LB agar plates.
For immunofluorescence microscopy, a protocol was used based on a previous study, 22

| Quantitative real-time PCR
The total RNA from each bacterial sample was isolated using the Trizol method described in a previous study. 23 Approximately

| Animal experiment
The acid environment of the stomach can be considered one of the host's first lines of defence against ingested bacteria. Prior to invasion, colonization and persistence within their host, Salmonella has evolved adaptive networks to protect themselves against the stress conditions. Therefore, whether the syringaldehyde planned for oral administration in this study has an effect on the acid stress response of S. Typhimurium remains to be verified. Before starting the animal experiment, bacteria survival test in artificially simulated gastric fluid (SGF) and relative mRNA transcription level of the acid resistance gene were performed according to the previous study. 24,25 The primers of targeted genes are shown in Table S1.

| Statistical analysis
The experimental data were assessed by unpaired two-tailed t tests using GraphPad Prism 5.0 (GraphPad software, La Jolla, CA) and P values are indicated as follows: **P < 0.01, *P < 0.05.

| Syringaldehyde inhibits the T3SS-mediated translocation of a SipA-lactamase fusion
To identify natural compounds capable of inhibiting translocation of the effector protein SipA into epithelial cells, a SipA-TEM-1-β-lactamase fusion reporter system was used as described previously. 2 We showed that the terpenoid compound syringaldehyde ( Figure 1A) was able to effectively inhibit the translocation of SipA into HeLa cells ( Figure 1B). The release of lactate dehydrogenase was de-

| Syringaldehyde inhibits T3SS effector protein expression through inhibition of SPI transcriptional regulation without affecting bacterial growth
We assessed whether syringaldehyde has an effect on the expression of T3SS effector proteins (SipA, SipB and SipC) via a Western blotting assay. As shown in Figure 2A Figure 2D). Taken together, these results showed that syringaldehyde inhibited the expression of effector proteins through SPI transcriptional regulation without affecting bacterial growth.

| Syringaldehyde inhibits S Typhimuriummediated invasion of HeLa cells
The above results indicated that syringaldehyde inhibits the expression of various Salmonella invasive proteins. Furthermore, we evaluated the inhibitory effects of syringaldehyde on the adhesion, invasion and replication of HeLa cells by S. Typhimurium SL1344 using a classic gentamicin protection assay. The results showed that syringaldehyde only inhibited the bacterial invasion of HeLa cells, meanwhile had no significant effect on bacterial adhesion and intracellular replication compared to the control group ( Figure S1A,B). The results shown are from one representative experiment performed in triplicate by LDH release assay. Significant differences between the syringaldehyde treatment and blank control groups are indicated (*P < 0.05, **P < 0.01) At a concentration of 0.18 mmol/L, the SL1344 invasion of HeLa cells was significantly inhibited, as the rate of bacterial internalization was reduced by 60.99 ± 0.05% ( Figure 3A). S. Typhimurium invasion was also assessed through an immunofluorescence analysis, which yielded the same results ( Figure 3B). These results demonstrated that the syringaldehyde treatment effectively blocked S.
Typhimurium invasion of HeLa cells.

| Syringaldehyde reduces S Typhimuriummediated HeLa cell injury
Previous studies have shown that S. Typhimurium SPI-1 mutant strains are unable to damage eukaryotic cells. 29 Therefore, we assessed the ability of syringaldehyde to protect HeLa cells from S.
Typhimurium-mediated damage. After co-culturing SL1344 and F I G U R E 4 Protective effects of syringaldehyde on S. Typhimurium-mediated mouse infection. A, Syringaldehyde prolongs the survival time and increases the survival rate of mice infected with S. Typhimurium. Wild-type S. Typhimurium SL1344 and invA mutant strains used for oral gavage infection of BALB/c female mice. A dose of 5 × 10 7 CFU bacteria of each strain was used to treat different groups of mice (n = 10), which were monitored for survival daily for 10 d after infection. B, Gross lesion observations of caecal tissues sections were prepared from each group. C, Histopathology observations of caecal tissue. The caecum tissues sections were prepared from each group and stained with haematoxylin and eosin. Finally, the caecal epithelial damage was evaluated by observations using an optical microscope. D, Syringaldehyde reduces the bacterial load in several organs of the infected mice. The bacterial load in the livers and spleens of mice was determined at 5 d after infection from tissue homogenate using the colony count method. The results shown are from three independent experiments performed in triplicate. E, In the syringaldehyde treatment group, cytokine production was reduced in the caecal tissue. All experiments were performed with caecal tissues from at least five mice and similar results were obtained from at least three independent experiments performed in triplicate. Error bars represent the standard errors of three datapoints. The level of significance is indicated as follows: NS, no significance, *P < 0.05; **P < 0.01. The P value was calculated by comparing the values to that of the blank control group  Figure 3C). Taken together, these results demonstrated that syringaldehyde can inhibit S. Typhimurium SL1344-mediated HeLa cell injury.

| Syringaldehyde increases the survival rate of mice infected with S Typhimurium
In To investigate the impact of the syringaldehyde treatment on the pathological manifestations of caecal injury, we performed histopathologic analyses of caeca from S. Typhimurium-infected mice that received 100 mg/kg of syringaldehyde or 0.9% NaCl as a control. As shown in Figure 4B, caeca from the mice without the syringaldehyde treatment after being infected with wild-type S.
Typhimurium SL1344 exhibited significant damage, whereas tissue from mice treated with syringaldehyde displayed slight damage and inflammation that were similar to that observed in the control mice treated with 0.9% NaCl and those infected with the invA mutant.
Notably, histopathological analyses showed that mice infected with wild-type S. Typhimurium displayed submucosal oedemas, reduced numbers of goblet cells and intestinal villi that were largely shed and ruptured, whereas through histopathological analyses, mice treated with syringaldehyde or infected with invA mutant only displayed slight submucosal oedemas and reduced numbers of goblet cells that was similar to that observed in the control mice treated with 0.9% NaCl ( Figure 4C). Similarly, syringaldehyde-treated group significantly reduced the amount of bacteria associated with such organs as liver and spleen, indicating reduced bacterial dissemination in syringaldehyde-treated mice ( Figure 4D). As expected, the caecal histopathological results were supported by the cytokine levels observed in these tissue samples, which were significantly lower in the syringaldehyde-treated group than in untreated mice ( Figure 4E). The effects of syringaldehyde on the ability of S.
Typhimurium to survive acid stress assay also showed that syringaldehyde had no significant effect on the acid resistance and the major acid resistance gene relative mRNA level of S. Typhimurium ( Figure S2), which further proved that the protective effect of syringaldehyde on Salmonella-infected mice was achieved by inhibiting T3SS. Taken together, our results showed that the syringaldehyde provided systemic protection against S. Typhimurium in a mouse infection model.

| D ISCUSS I ON
At present, the frequent occurrence of pathogens that are resistant to traditional antibiotics poses a great threat to public health, especially for developing countries with weak public health programs.
Since the beginning of the 1990s, Salmonella strains exhibiting mul- Syringaldehyde is a traditional Chinese medicine obtained from a wide range of sources, having been identified and isolated from the stems of Hibiscus taiwanensis and others plants. As a terpenoid component of plant volatile oil, syringaldehyde (3,5-dimethoxy-4-hydroxybenzaldehyde), has been shown to have hyperglycemic, 37 anti-oxidative and anti-apoptotic activities. 38 In addition, T3SS is conserved in many Gram-negative pathogens, we showed that syringaldehyde is a potential inhibitor for developing natural small molecule compounds against bacterial pathogenesis in vitro and in vivo, further demonstrating that targeting the T3SS is a practical strategy. In summary, syringaldehyde may be an ideal leading compound with broad application to treat Salmonella infections in the future.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science

Foundation of China (31620103918) and the Natural Science
Foundation of Jilin Province (NO. 20170520006JH for XC).

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.