Inhibition of Salmonella enterica serovar Typhimurium Type (cid:0) Secretion System and Infection Using Small Molecule Quercitrin

Aims This study was conducted to screen the type (cid:0) secretion system (T3SS) inhibitors of Salmonella enterica serovar Typhimurium (S. Typhimurium) from natural compounds. Through systemic analysis the pharmacological activity and action mechanism of candidate compounds in vivo and in vitro. Methods and results Using an effector-β-lactamase fusion reporter system in S. Typhimurium, we discovered that quercitrin could block effector SipA translocation into eukaryotic host cell without affecting bacterial growth, and inhibit invasion or epithelial cells damage. Using β-galactosidase activity and Western blot assay, it was found that quercitrin signicantly inhibits the expression of SPI-1 genes (hilA and sopA) and effectors (SipA and SipC). The animal experiment results indicated that quercitrin reduces mortality, pathological damages and colony colonization of infected mice.


Introduction
Salmonella is a Gram-negative enterobacter commonly found in external environment, that also exists in the intestinal tract of humans and animals 1 . Salmonella has a wide range of hosts, mainly cause animal diseases such as chicken, pigs, horses, cattle and sheep, and cause contamination of animal food. It mainly causes human diseases such as typhoid, paratyphoid and gastroenteritis through the faecal-oral pathway 2 . Salmonellosis is one of the most common food-borne diseases in humans 3 . Typhoid fever caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) remains a signi cant human health problem, especially in developing countries 3 . In short, Salmonella not only hinders the development of the livestocks industry and endangers food safety, but also causes panic about public health safety issues.
Antibiotics are still the rst choice against Salmonella .spp infections. However, antibiotic resistance leads to a huge challenge to preventing the spreading of infection disease of Salmonella 4 . New antiinfection therapies are urgently needed, such as using alternative drugs to prevent or treat pathogens infections. S. Typhimurium induces in ammatory diarrhoea and invades non-phagocytic epithelial cells using the type secretion system (T3SS) encoded by SPI-1 5 , which is critical to bacteria infection. The T3SS is a needle-like structure that injects several effectors into eukaryotic host cytoplasm 6 . Structural genes for the functional T3SS device assembling and effectors are encoded in the SPI-1 prg/org, inv/spa, and sic/sip operons, meanwhile three AraC-like regulators, hilD, hilC, and rtsA, control the expression of hilA in SPI-1 signal caccade pathway activation 7 . HilD is the dominant regulator, while HilC and RtsA act as the signal ampli ers 8 . SipA could bind to host cells actin and promote bacterial internalization 9 . The killing effect of macrophages is caused by Salmonella-induced apoptosis and occurs after caspase-1 is activated by SipB 10 . SipC is responsible for the translocation of effectors and regulation of actin 11 . Thus, T3SS has become an attractive drug screening target to reduce the risk of antimicrobial resistance iby indirect sterilization or antimicrobial dependence manner while treating bacterial infection.
In this study, quercitrin, a glycosylated avonoid isolated from whole grass of Hypericum perforatum, which could signi cantly inhibit the biological function of Salmonella T3SS. In addition, its pharmacological activity in vitro was evaluated by eukaryotic cell adhesion, invasion and cell damage assay, and the protective effect was con rmed in vivo. In conclusion, quercitrin could be used as a potneial drug for prevention and treatment of Salmonella infection.

Materials And Methods
Bacterial strains, growth conditions, and natural compounds The wild-type S. Typhimurium SL1344 was donated by Professor Xiaoyun Liu of Peking University. In accordance with previous study, InvA is a prominent inner-membrane component of the T3SS apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria 12 . We used invA mutant as negative control involved in encoding key structural proteins of T3SS-1. The hilA::lacZ (JS749) and sopA::lacZ (JS751) strains were provided by Dr James Slauch from University of Illinois. S. Typhimurium SL1344-SipA-3×Flag is produced by inserting PKS 3×Flag into chromosome 13 . Bacteria were stored at -80°C in Luria-Bertani (LB) broth containing 40% glycerol and overnight cultures grown in LB broth at 37°C with aeration. All used natural compounds including quercitrin are from the candidate compounds libraries constructed and preserved in our laboratory, which are purchased from Heibpurify (Chengdu, China). Unless otherwise speci ed, it is generally prepared at 40 mg/ml of dimethyl sulfoxide (DMSO) solution and stored at 4°C.
High-throughput screening for natural compounds T3SS inhibitors by effector-β-lactamase fusion reporter system HeLa cells were plated into 96-well plates at a density of 1.2×10 4 cells/well and incubated overnight before infection. S. Typhimurium SL1344 containing SipA-β-lactamase fusion plasmid and the invA mutant grown overnight at 37°C in LB (0.3 M NaCl to activate T3SS) and diluted by 1:20 in the presence of quercitrin with DMSO as controls. After incubation for 4 h at 37℃ with shaking, the bacterial suspensions were adjusted to 3×10 6 CFUs/ml. 200 µl of each sample was transferred into a 96-well plate, and the monolayers were infected at a multiplicity of infection (MOI) of ~50 for 1 h. Non-internalized bacteria were washed three times with PBS, and incubated with 100 µl PBS containing 6×CCF4/AM reagent (Life Technologies, USA) for 45 min at room temperature. Fluorescence micrographs were captured and analyzed by uorescence microscope (model: Olympus IX-81, Japan).

Determination of bacterial viability following quercitrin exposure
In order to identi cation the potential bactericidal or bacteriostatic effects of quercitrin, bacterial viability following quercitrin exposure determination was performed. Overnight S. Typhimurium SL1344 cultures were diluted 1:100 in LB for 2 h at 37°C until the optical density of 600 nm (OD 600nm ) reached logarithmic growth phase. The culture contained different concentrations of quercitrin, and the group without quercitrin was the positive control. The OD 600nm were measured with a UV-2100 spectrophotometer (Unico, China) every 30 min until the stationary phase.

Cytotoxicity of quercitrin indicated by lactate dehydrogenase release
The HeLa cells were cultured in Dulbecco's Modi ed Eagle's Medium/high glucose (DMEM, HyClone, USA) containing 10% foetal calf serum (Biological Industries, Israel), and 1% penicillin-streptomycin (MRC, USA) at 37℃ and containing 5% CO 2 for more than 24 h. The cells were inoculated into 96-well plates at a concentration of 1×10 5 cells/ml and allowed to adhere overnight. Adherent HeLa cells were subjected to 8 h of quercitrin exposure (at concentration of 0~64 µg/ml) in triplicate. DMSO (0.1% of nal concentration) and 0.2% Triton X-100 were set as the negative or positive controls. The lactate dehydrogenase (LDH) release measured by the LDH cytotoxicity detection kit (Roche, Germany) and microplate spectrophotometer (Tecan, Austria) at a wavelength of 490 nm 14 .

Adherence assay
In brief, HeLa cells were infected with S. Typhimurium at an MOI of 50. After incubation at 37°C for 20 min, the wells were washed three times to remove unattached bacteria. Then, the cells were lysed with 0.2% (v/v) Triton X-100 for 10 min and plated on LB agar plates.

Gentamicin protection and immuno uorescence internalization
The effect of quercitrin on bacterial invasion was determined by gentamicin protection with some modi cations as described previously 15 . HeLa cells (4×10 4 cells/well) were plated in 24-well plates, and overnight cultures of S. Typhimurium SL1344 were added to fresh LB containing different nal concentrations of quercitrin for 4 h. HeLa cells were infected at an MOI of ~100 at 37°C in 5% CO 2 for 1 h.
HeLa cells were washed three times with PBS to remove unattached bacteria, and then incubated in DMEM containing gentamicin (100 µg/ml) at 37°C for 5 min. After washing three times with PBS, cells were lysed in 0.2% (v/v) Triton X-100 to enumerate viable intracellular bacteria through a series of dilutions on LB agar plates.
For immuno uorescence analysis, infected cells on coverslips were xed with 4% paraformaldehyde for 10 min at room temperature. After blocking for 1h, the cells were incubated with 1000-fold diluted primary antibody of S. Typhimurium for 1 h, and 1000-fold dilution of Alexa Fluor 488-conjugated secondary antibody (Abcam, USA) for 30 min to stain extracellular bacteria. Then, 0.3% (v/v) Triton X-100 was used to permeabilize cells for 10 min, and incubated in Alexa Fluor 594-conjugated secondary antibody (Abcam, USA) diluted 500-fold for 30 min to stain the bacteria in the cells. Finally, coverslips were mounted on slides with DAPI (Abcam, USA) for 10 min. Cell coverslips were visualized using a uorescence microscope (Olympus IX-81, Japan).

Antibodies and western blot
Western blot analysis was used to further validate the effect of quercitrin on expression of T3SS genes and effector. Brie y, the overnight cultures of SL1344, SipA-3×Flag-SL1344 and SipB-3×Flag-SL1344 were added to fresh LB containing different concentrations of quercitrin. Each group was centrifuged at 12000 rpm for 10 min and the precipitates were collected. Resuspend the bacteria in 100µl 1×loading buffer, and boil them at 95°C for 6 min. Protein samples were separated using sodium dodecyl sulfate β-Galactosidase activity assay β-Galactosidase activity was determined using a previously described method 16 . Brie y, overnight cultures of JS749 and JS751 bacterial were added to fresh LB with or without quercitrin. Centrifuge the culture at 12000 rpm for 10 min and resuspend it in Z-buffer. Then added 20 µl 0.1% SDS and 40 µl chloroform to bacteria suspension, shake and lyse for 30 s.
Add 100 µl mixture into a 96-well plate with 3 replicates in each group. Add 20µl ONPG to initiate the reaction, incubated at room temperature for 10 min, and nally 50µl 1 M Na 2 CO 3 to terminate the reaction. The absorbance was detected at wavelength of 450 nm with a microplate reader (Tecan, Austria).

Animal experiment
All experimental protocols were reviewed and approved by the Animal Welfare and Research Ethics Committee at Jilin University (2021052625M). All animal experiments were performed in strict compliance with the guide-lines of the Animal Welfare Council of China. All eforts were made to minimize the sufering of animals, and daily health checks were performed throughout the experiments. And all the procedures were carried out in accordance with ARRIVE guidelines.
In order to reduce the intestinal ora, we added 5 g/L streptomycin to the drinking water three days before the experiment 16 . The model of S. Typhimurium infection was established by intragastric inoculation. The mice were divided into three groups with 10 mice in each group, including 50 mg/ml quercitrin treatment group, PBS control group and DMSO challenge group. Brie y, fasting and banning water for 8 h before infection, mice with 5×10 7 or 10 7 CFU, test survival rate and other indicators, gavage once every 12 h for 4 days. The treatment group was given 50 mg/kg quercitrin, the control group was given the same amount of PBS. Mice in each group were killed by cervical dislocation. Liver, spleen and cecum tissues were removed by aseptic operation. The organ was weighed, ground evenly with 0.2% Triton X-100, were serially diluted and plated on LB plates containing streptomysin, cultured overnight at 37℃ and colonies were counted the next day. Pathological tissue sections were prepared with hematoxylin and eosin (H&E) and histopathology was performed by capturing images using an optical microscope.

Data analysis
All experiments data were conducted with least three biological replicates for analysis and statistical signi cance testing by using GraphPad Prism 6.0 software (La Jolla, CA). Results from treated and control samples were expressed as the mean ± SEM and analyzed using Student's t tests analysis. *P < 0.05; **P < 0.01; NS, P > 0.05, not signi cant.

Screening natural compound inhibitors of S. Typhimurium T3SS
Seven inhibitors of the virulence factor T3SS were identi ed from 354 natural compounds by using the effector-lactamase fusion reporter system in Salmonella typhimurium, as in previous studies 17 . At the same time, we monitored the LDH release rate in HeLa cells after 6 hours of incubation, eliminating one compound that had a negative impact on cell viability. Then, Western Blot and β-lactamase colorimetry were used to screen quercitrin inhibiting T3SS (Figure 1).

Quercetin blocks the translocation of T3SS effector sipA
Quercitrin was screened to effectively block the translocation of SipA, the T3SS effector of S. Typhimurium (Figure 2A). In order to verify that the mechanism of the inhibitory activity without bactericidal or bacteriostatic, the growth curve showed that quercitrin did not inhibit the activity of bacteria in the concentration range of 4 µg/ml~32 µg/ml( Figure 2B). Our screen of natural compounds and uorescence images showed that 16 µg/ml quercitrin signi cantly inhibited the translocation of T3SS effector SipA into eukaryotic host cells compared with the control group( Figure 2C).

Quercitrin inhibits S. Typhimurium adhesion of eukaryotic host cells in a dose-dependent manner
We then tested the inhibition ability of quercitrin to inhibit S. Typhimurium adhesion to cultured eukaryotic host cells 18 . Before the adhesion assay, the cytotoxicity of quercitrin to HeLa cells were determined by Lactate dehydrogenase (LDH) release assay. HeLa cells were co-incubated with different concentrations of quercitrin, but no cytotoxicity was observed in the concentration range of 2 µg/ml~64 µg/ml( Figure   3A). Next, bacteria cultured in the presence of quercitrin showed signi cant reduction in adhesion of HeLa cells in a dose-dependent manner. The adhesion inhibitory effect of quercitrin at 32 µg/ml was reduced to 15% compared to untreated group( Figure 3B).

Quercitrin inhibits S. Typhimurium invision of eukaryotic host cells in a dose-dependent manner
As an intracellular bacterium, adhesion and invasion are prerequisites for bacteria-mediated eukaryotic host cell damage. 32µg/ml of quercitrin reduced S. Typhimurium-mediated cell damage by more than 80%( Figure 4A). The protective effect of quercitrin in HeLa cells damage was reduced up to 50% compared with untreated group( Figure 4B). As expected, the inhibition of S. Typhimurium invasion could be readily observed by immuno uorescence analysis of intracellular and extracellular bacteria( Figure 4C). These results demonstrated that quercitrin inhibit the T3SS-dependent S. Typhimurium adhesion and invasion of eukaryotic host cells without cytotoxicity.

Quercitrin inhibits expression of T3SS related genes and effectors in S. Typhimurium
According to the above results, quercitrin inhibited the translocation of T3SS effector SipA. Using Western Blot with chromosomal insertions of 3×Flag and wild-type SL1344 strain to prove whether quercitrin inhibited the expression of effectors. Indeed, we found that quercitrin signi cantly inhibited the expression of SipA and SipC, it was dose-dependent within a certain range compared with internal control ICDH( Figure 5A). Compared with the untreated group, the inhibition rates of 16 µg/ml quercitrin to SipA-3×Flag was more than 50%, and the expression of SipC, another effector responsible for translocation and actin regulation 11 , was reduced more than 30% after co-culture with 16 µg/ml quercitrin( Figure 5B). The western blot results showed that quercitrin inhibited the T3SS effectors expression of SipA and SipC. Other important effectors were further detected by β-galactosidase activity assay. The result shown that quercitrin signi cantly reduced the expression of hilA and sopA, which are mainly responsible for the regulation and function of S. Typhimurium T3SS( Figure 5C~D).

Quercitrin reduces S. Typhimurium infection in mice
The streptomycin-pretreated of S. Typhimurium infection mouse model was established according to the previously research 19 . Mice were infected with the S. Typhimurium SL1344 by oral gavage at the dose of 1×10 7 CFUs, and control group was set without treatment. The results shown that the mortality was 80% on the 5 day, until all mice died on the 7 day. In contrast, the mortality of quercitrin treatment group was 20% until the 7 day ( Figure 6A). Quercitrin not only prolonged the survival time of mice infected with S. Typhimurium, but also improves the survival rate of infected mice. At the same time, the macropathological changes and analysis of HE staining in cecal tissues were performed as previously described 20 . Compared with the infected group, colony colonization in liver, spleen and cecum in quercitrin treatment group was signi cantly reduced ( Figure 6B). The S. Typhimurium challenged group had severe cecal bleeding, and the blind end was signi cantly atrophied. There was no signi cant difference between the control group and the quercitrin treatment group, suggesting that quercitrin signi cantly reduced the intestinal damage of S. Typhimurium infection( Figure 6C). The histopathological analysis revealed severe submucosal edema, a loss of goblet cells, impaired epithelial integrity, and the in ltration of polymorphonuclear cells (PMN) in the lamina propria of the wild-type infection group. In contrast, the quercitrin-treated group showed mild intestinal damage and in ammation( Figure 6D). In summary, quercitrin effectively protects mice from S. Typhimurium infection.

Discussion
Although antibodies and vaccines against pathogens infections are further along in the pipeline, long development cycles and high economic costs prevent them from reaching the market. In recent years, antibiotic alternative strategies targeting pathogen virulence factors have shown promise. Because virulence factors, but not the growth, of many Gram-negative bacterial pathogens depends on the multicomponent type three secretion system injectisome (T3SSi), the T3SSi has been an attractive target for identifying novel drugs that inhibit its function to render the pathogen avirulent.
T3SS is widely distributed in Gram-negative bacteria, and its main function is to secrete and transport pathogenic bacteria virulence factors 21 . Inhibition of Salmonella T3SS can achieve anti-infection effect. Previous studies have reported that screening inhibitors targeting Salmonella T3SS have achieved signi cant progress. For example, salicylidene acylhydrazides (SAHs) are the rst identi ed and most widely studied class of synthetic small molecule inhibitor that target the T3SSi across many bacterial species 22 . Lico avonol exerts a strong inhibitory effect on the secretion of the S. Typhimurium SPI-1 effectors by regulating the transcription of the SicA/InvF genes and the transport of the SipC 23 . According to Zhang et al. study, thymol was capable of blocking the activity of T3SS-1 at concentrations that did not affect either bacterial viability or the integrity of mammalian cell membranes. Although the detection methods are constantly updated, the scarcity of natural product origin inhibitors and the unclear mechanism of action still limit the discovery and clinical application of natural compounds or derivatives 17 .
Compared with conventional antibacterial drugs, antibodies and probiotics, most of these candidate drugs inhibit virulence factors that are not essential for bacteria, and the possibility of inducing bacterial resistance is signi cantly reduced 24 . Yet importantly, study of such a molecule could help elucidate structure-function relations of the pathogen T3SS and serve as a highly e cient molecular platform to develop active molecules against homologous components in other T3SS 25 .
Quercitrin is a natural glycosylated avonoid obtained from the whole grass of Hypericum perforatum and the bark of different species of oak trees with anti-bacterial, anti-in ammatory and hypidemia effects 26 . In this study, quercitrin, a T3SS inhibitor, was screened from a series of natural compounds by SipA-TEM-β-lactamase fusion reporter system. As the results shown that quercitrin inhibited S. Typhimurium invasion of host cells and host cell damage without affecting bacterial growth. Animal experiments showed that quercitrin reduced the mortality and pathological damage caused by S. Typhimurium infection. In conclusion, quercitrin is an ideal leading compound for treatment of S. typhimurium infection and has broad application prospects in the future. Figure 1 Flowchart for high-throughput screening of S. Typhimurium T3SS inhibitors.    Quercitrin inhibits the invasion of host cells by S. Typhimurium. (A) Inhibitory effect of quercitrin on the invasion of S. Typhimurium. HeLa cells were infected with S. Typhimurium SL1344 pretreated with quercitrin. (B) Quercitrin protects HeLa cells from S. Typhimurium mediated injury. The LDH release data showed that the degree of cell damage caused by S. Typhimurium was signi cantly reduced after pretreatment with quercitrin.All data are shown as the mean ± SEM from three independent experiments.

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