Establishment of a STING-Deficient HepG2 Cell Line through CRISPR/Cas9 System and Evaluation of Its Effects on Salmonella Replication

Background Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a common food-borne pathogen that causes gastroenteritis and can lead to life-threatening systemic disease when it spreads to vital organs, such as the liver. Stimulator of interferon genes (STING) is a crucial regulator of the host's innate immune response to viral infections, while its role in bacterial infections remains controversial. This study aims to establish a STING-deficient HepG2 cell line through the CRISPR/Cas9 system and evaluate its effects on Salmonella replication. Methods In this study, a STING knockout HepG2 cell line was constructed through the application of CRISPR/Cas9 technology. We assessed cell viability and proliferation using the CCK-8 assay. Subsequently, we investigated the effect of STING deletion on Salmonella replication and the expression of type I interferon-related genes. Results The STING knockout HepG2 cell line was successfully constructed using the CRISPR/Cas9 system. The proliferation capability was diminished in STING-deficient HepG2 cells, while Salmonella Typhimurium replication in these cells was augmented compared to the wild-type (WT) group. Following Salmonella infection, the transcriptional responses of type I interferon-related genes, such as IFNB1 and ISG15, were inhibited in STING-deficient HepG2 cells. Conclusions We successfully constructed a STING-deficient cell line. Our finding of increased Salmonella Typhimurium replication in STING-deficient HepG2 cells provides the basis for further studies on pathogen-host interactions.


Introduction
Salmonella infections are prevalent globally and continue to pose signifcant public health challenges in many developing nations.Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a Gram-negative, facultative intracellular bacterial pathogen that can lead to severe gastroenteritis and systemic infammation in humans and animals [1].Additionally, it serves as a widely used model organism to investigate the molecular mechanisms involved in bacterial pathogenesis and pathogen-host interactions.As one of the most widespread pathogens transmitted through food and water, Salmonella Typhimurium often infltrates the gastrointestinal barrier, resulting in symptoms like diarrhea and acute infammation.Later in the infection process, it can disseminate to the mesenteric lymph nodes and may even spread to extraintestinal tissues, such as the liver [2].Severe hepatic involvement with an acute hepatitislike clinical appearance has been reported as a possible consequence of Salmonella infection [3,4].However, the molecular mechanisms of how hepatocytes respond to Salmonella infection remain incompletely defned.
Type I interferons (IFNs) are involved in the host response to Salmonella infection.Autophagy is a signifcant antimicrobial response that inhibits Salmonella growth [5].IFN-β can be induced by autophagy through Toll-like receptors (TLRs) and the TRIF pathway, providing a protective efect on the host [6].However, another study found that IFN-β increased host susceptibility to infection by restricting innate immune responses [7].Besides autophagy, stimulator of interferon genes (STING) is one of the main activators of IFN-β production.Te rapid detection of microbial agents is crucial to efectively trigger the host's defense mechanisms against infection.STING is an endoplasmic reticulum (ER)-associated membrane protein known to trigger type I IFN and proinfammatory responses upon the detection of cytosolic DNA by cyclic GMP-AMP synthase (cGAS) [8].Te cGAS-STING pathway is an important cytosolic surveillance pathway (CSP), and increasing evidence demonstrates its essential role in host defense against a variety of bacterial pathogens [9][10][11][12].Moreover, STING is also a direct sensor of cyclic dinucleotides (CDNs) generated by numerous intracellular bacteria [13].Although the cGAS-STING pathway has been extensively studied for its role in promoting the antiviral innate immune response, its function in bacterial infections remains debatable.Terefore, constructing STING knockout cell lines is helpful for clarifying the role of STING in Salmonella infection.
HEK293T and Vero-E6 cells are known to have defciencies in cGAS-STING signaling [14].We aimed to investigate the cellular response to Salmonella infection in both STING-containing and STING-defcient systems, focusing on hepatocytes.Terefore, we set out to delete STING in cells that have demonstrated STING activation through gene knockout.As previously reported, STING is not expressed in primary human hepatocytes and Huh7 cells [15].However, researchers have detected STING expression and cGAS-STING signaling activation in another human hepatocyte-derived cell line, HepG2 [15,16].
Te clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a recently developed and highly efcient genome editing technology [17].Guided by gRNA, Cas nucleases orchestrate the precise cleavage of DNA double strands, playing a crucial role in gene editing.Tis method ofers benefts such as convenience, efciency, cost-efectiveness, and ease of use [18].In this study, we constructed a STING knockout HepG2 cell line using CRISPR/Cas9 technology.Subsequently, we investigated the efect of STING deletion on Salmonella replication.Te generated knockout cell line represents an optimal resource for subsequent investigations into Salmonella infection.

Bacterial Strain and Infection. Salmonella
Typhimurium strain SL1344 in this study was kindly provided by Professor Qian Yang (Nanjing Agricultural University, Nanjing, China).Salmonella Typhimurium strain was grown in Luria Bertani (LB) medium at 37 °C with shaking overnight.Te following day, it was diluted at a ratio of 1 :100 with fresh LB medium and then cultured until it reached the logarithmic phase of growth.Upon washing 3 times with sterile PBS, bacterial numbers were estimated and adjusted based on the optical density at 600 nm absorbance and ready for the further experiments.For infection, bacteria were washed in PBS and subsequently resuspended in DMEM at a multiplicity of infection (MOI) of 10.

Construction of sgRNA
Plasmid.DNA fragment containing the U6 promoter and sgRNAs was amplifed by polymerase chain reaction (PCR) using PUC57-sgRNA-U6 plasmid as a template with the primers in Table 2. Ten, PCR products and primary pGL3-U6-2sgRNAs plasmids were digested with Esp3I enzyme (ER0451; Termo Scientifc) and ligated with T4 DNA ligase (2011A; Takara) according to manufacturer's instructions.

Construction of STING Knockout Monoclonal Cell Line.
Cells were transfected with constructed pGL3-U6-2 sgRNAs together with pSt1374-N-NLS-Flag-Cas9 plasmids using the Lipofectamine ™ 3000 Transfection System (L3000001; Termo Scientifc) according to manufacturer's instructions.After 48 hours of transfection, cell screening was conducted using a puromycin concentration of 2 μg/mL.Te residual cell population was subjected to single-cell separation using a limited dilution method and transferred into a 96-well plate.Monoclonal identifcation was performed after 2 weeks of cell culture.

PCR and Sanger Sequencing of STING Knockout Cell Line.
Monoclonal cells were validated through PCR followed by Sanger sequencing.Total cellular DNA was extracted using FastPure Cell/Tissue DNA Isolation Mini Kit (DC102; Vazyme).PCR reaction was performed according to the protocol of 2 × Phanta Max Master Mix (P515; Vazyme) with the primers in Table 3. Ten, the PCR reaction settings were set up as follows: initial denaturation 95 °C for 3 min, 30 cycles of denaturation 95 °C for 15 s, annealing 55 °C for 15 s, 72 °C extension for 1 min, and 72 °C extension for 5 min.Next, the PCR product was purifed using electrophoresis and FastPure Gel DNA Extraction Mini Kit (DC301; Vazyme).Sanger sequencing was performed at GENEWIZ Co., Ltd.(Suzhou, China).2.9.Bacterial Replication Analysis.For the assessment of bacterial replication, 4 × 10 5 HepG2 cells were initially seeded into 12 well plates and then infected with SL1344 at an MOI of 10 the next day.Te amount of bacterial suspension added to the cell cultures was calculated from growth curves measured in the laboratory before.After a 0.5 h infection, the cells were washed with PBS and subsequently cultured for an additional 0.5 h in DMEM-FBS (10%) supplemented with 100 μg/mL gentamicin (A620217; Sangon Biotech) to remove extracellular bacteria.Ten, cells were lysed with 0.3% Triton X-100 (V900502; Sigma-Aldrich), diluted cell lysates were plated onto SS agar culture plates, and the cultures were incubated at 37 °C overnight to enumerate the internalized bacteria [20].

RNA Isolation and Quantitative Real-Time PCR (qPCR).
Cells were lysed, and total RNA was extracted using TRIzol reagent.RNA was reverse transcribed by RT Reagent Kit (RR047A; Takara).qPCR was performed using SYBR Green Supermix kit (1725121; BIO-RAD) on CFX96 Touch Real-Time PCR Detection System (BIO-RAD).Primers are shown in Table 4.

Statistical Analysis.
Te analysis of experimental data was carried out by GraphPad Prism software (La Jolla, CA, USA) and IBM SPSS Statistics for Windows (Armonk, NY, USA).Statistical comparisons between two datasets were executed utilizing the unpaired Student's t-test.One representative dataset of at least three independent experiments was shown as the mean ± standard error of the mean (SEM).

Construction of STING Knockout HepG2 Cell Strain.
pGL3-U6-2sgRNAs plasmids and the PCR products containing the U6 promoter and sgRNAs were digested by Esp3I enzyme (Figures 1(a), 1(b)).After ligation, the combinant plasmid containing two sgRNAs targeting exon 6 of the STING gene was constructed (Figure 1(c)).Te recombinant and Cas9 expression plasmids were cotransfected into HepG2 cells, followed by puromycin screening and single cell separation.After monoclonal cell formation, total DNA of the cells was isolated, and PCR reaction was performed with primers designed on the exon 6 of human STING (Table 3).Sanger sequencing of the PCR products revealed that 38 nucleotides of exon 6 were missing, leading to a frameshift mutation in human STING, indicating that STING gene was knocked out successfully (Figure 1(d)).
When passaged at a proportion of 1 : 3, both STING knockout and WT cell lines achieved 90% confuence within 3 days.Western blotting analysis showed that protein levels were undetectable in the STING knockout cell line compared to the WT cell line, further confrming the efciency of the gene knockout (Figure 1(e)).

STING Knockout Suppresses HepG2 Cell Proliferation.
We then used the CCK-8 kit to examine how STING knockout afects the proliferation of the HepG2 cell line.Our results showed that the optical density (O.D.) values of the STING knockout group were signifcantly lower compared to the WT group at several time points after incubation with the CCK-8 reagent (Figure 2).Tis fnding indicates that STING knockout inhibits the proliferation ability of HepG2 cells.

STING Knockout Promotes Salmonella Replication.
To investigate the efect of STING knockout on Salmonella replication in the HepG2 cell line, internalized bacteria numbers was analyzed after infection at an MOI of 10 for 1 h.Te results revealed that Salmonella replication in the HepG2 cells with STING knockout was signifcantly higher compared to the WTgroup (Figure 3).Tis fnding indicates that STING plays a negative role in Salmonella replication.

Discussion
Salmonella Typhimurium is a bacterial pathogen that causes various diseases in humans and animals.In severe cases of Salmonella Typhimurium infection, the bacteria can enter the bloodstream (bacteremia) and spread to various organs, including the liver.Tis can lead to disseminated infection and systemic symptoms [1].STING is primarily known for its role in regulating the immune response against viral infections and certain intracellular pathogens.Some studies suggest that STING may also play a role in controlling bacterial infections by modulating infammation and immune responses [8].However, the interaction between Salmonella Typhimurium-a food-borne bacterial pathogen-and STING has not been studied as extensively as its interaction with viruses, particularly in nonphagocytic cells such as hepatocytes.Among common hepatic cell lines, HepG2 has been used as an in vitro model to investigate the cellular and molecular mechanisms involved in Salmonella infection and its impact on hepatocytes [22,23].Additionally, while STING expression is not detected in mouse and human primary hepatocytes, it is expressed and functional in HepG2 cells [15,16].In this study, we generated a STING-defcient HepG2 cell line using the CRISPR/Cas9 system, an efcient and widely used tool for generating knockout cell lines.We found that STING knockout cells are more susceptible to Salmonella infection, with increased bacterial growth.Tis indicates STING exerts an antibacterial efect in HepG2 cells.Chronic infammation resulting from persistent activation of the cGAS-STING pathway is strongly associated with cancer progression [24,25].In hepatocellular carcinoma tissues, the mRNA levels of genes involved in the cGAS-STING pathway are up-regulated [16].HepG2 is an immortal cell line derived from a hepatocellular carcinoma patient, and chromosomal instability (CIN) is a hallmark of human cancer [26].In our study, we found that STING knockout inhibited the proliferation ability of HepG2 cells.Te reduced cellular viability observed in HepG2 cells following STING deletion aligns with recent research, which suggests that the cGAS-STING pathway drives the survival of chromosomally instable cancers [27].One potential mechanism by which STING may suppress Salmonella Typhimurium replication is through the production of type I interferons and proinfammatory cytokines.Te role of IFNβ in Salmonella infection remains ambiguous.In macrophages, IFN-β has been shown to both promote and inhibit innate immune responses, such as the release of infammatory cytokines following Salmonella infection [6,7].Upon sensing bacterial DNA, STING can activate the production of interferons, which initiates an antiviral response [28,29].Although Salmonella Typhimurium is a bacterium, the antiviral response may still contribute to immune defense against pathogens.Our data showed that the mRNA level of IFNB1 was reduced in STING knockout cells, suggesting an antibacterial function of IFN-β in the HepG2 cell model of Salmonella infection.Further experiments are needed to manipulate IFN-β levels in this model to confrm its antibacterial role.ISG15 is an interferon-induced protein that aids in clearing invading bacteria [30].We observed a signifcant decrease in ISG15 mRNA expression in the STING knockout group, combined with increased Salmonella replication, suggesting that ISG15 may have an anti-infective role in HepG2 cells.In addition to type I IFN signaling, bacterial infection can directly activate ISG15 expression through the STING pathway [31].Terefore, STING deletion alone in Salmonella-infected HepG2 cells may signifcantly contribute to the downregulation of ISG15 expression.Another possible pathway involves the interaction between STING and pattern recognition receptors (PRRs), such as TLRs [32,33].Activation of TLR can enhance STING signaling pathway, potentially boosting host immune responses [34].During Salmonella infection, autophagy plays a critical role in inhibiting bacterial survival [35].STING has been reported to directly activate autophagy to regulate the innate immune response [36,37].Tus, autophagy may be involved in STING-mediated restriction of Salmonella survival in HepG2 cells.Te detailed mechanisms underlying our fndings warrant further investigation.
In conclusion, we constructed a STING knockout HepG2 cell line using the CRISPR/Cas9 system.Our fndings reveal   Polymerase chain reaction PRR: Pattern recognition receptor SEM: Standard error of the mean STING: Stimulator of interferon genes TLR: Toll-like receptor WGE: Wellcome Sanger Institute Genome Editing WT: Wild-type.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :
Figure 1: Construction of STING knockout HepG2 cell strain.(a) Agarose gel electrophoresis of isolated plasmids, including nondigested primary pGL3-U6-2sgRNAs plasmids (control) and plasmids digested by Esp3I restriction enzyme (digested).(b) Agarose gel electrophoresis of PCR products containing the U6 promoter and sgRNAs targeting exon 6 of human STING gene and digested by Esp3I restriction enzyme.(c) Constructed pGL3-U6-2sgRNAs plasmid, sgRNA, and scafold sequence located downstream of the U6 promoter, and puromycin coding sequence located downstream of EF-1 alpha promoter.(d) DNA sequence analysis of WT and knockout cell line showing the STING mutation, the red box indicates the deleted 38 base pairs.(e) Western blotting analysis of STING protein in WT and STING knockout (KO) HepG2 cell lines.
Analysis.Te total protein of WT or STING knockout HepG2 cells were extracted using RIPA Lysis Bufer (R0278; Sigma Aldrich) containing the Protease 2.8.Cell Proliferation Analysis.CCK-8 Cell Counting Kit (A311; Vazyme) was used to perform cell proliferation assays.In this procedure, 2 × 10 4 HepG2 WT or STING knockout cells were seeded in 96 well plates and added with 10 μL of the CCK-8 reagent for another 1 h, 2 h, 3 h, and 4 h.Optical density was measured at 450 nm.

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Journal of Pathogens that STING deletion inhibited HepG2 cell proliferation and facilitated Salmonella Typhimurium replication.It is important to note that our understanding of STING's role in bacterial infections, including Salmonella, is still developing.Te established cell line serves as an efective tool for further research on Salmonella infection.