LncRNA SBF2-AS1 Regulates Pyroptosis to Promote Chlamydia trachomatis Growth Through miR-196b-5p/RIPK2 Axis

TL;DR

This study shows how the bacteria Chlamydia trachomatis uses a long non-coding RNA to block cell death, helping it survive and multiply inside host cells.

Contribution

The discovery of a novel lncRNA-mediated mechanism by which Chlamydia evades pyroptosis through the miR-196b-5p/RIPK2 axis.

Findings

SBF2-AS1 is upregulated during Chlamydia infection and inhibits pyroptosis.

SBF2-AS1 acts as a competing RNA for miR-196b-5p, which targets RIPK2.

Disrupting SBF2-AS1 or RIPK2 triggers pyroptosis and suppresses Chlamydia replication.

Abstract

Pyroptosis enables host cells to eliminate intracellular pathogens effectively. However, how Chlamydia trachomatis (C. trachomatis) evades host pyroptosis remains unclear. This study reveals that C. trachomatis exploits the host Long non-coding RNA (lncRNA) SBF2-AS1 as a key factor to regulate the host pyroptosis. The SBF2-AS1 was significantly upregulated during C. trachomatis infection. Knockdown of SBF2-AS1 activated NLRP3/caspase-1/GSDMD pyroptosis pathway. Mechanistically, it verified that SBF2-AS1 functions as a competing endogenous RNA for miR-196b-5p targeting RIPK2 through dual-luciferase reporter gene assay. We further identified the interaction between RIPK2 and Caspase-1 by Co-immunoprecipitation (Co-IP). Silencing SBF2-AS1 or RIPK2, as well as overexpressing miR-196b-5p, triggered pyroptosis and suppressed the replication of C. trachomatis. In conclusion, C. trachomatis upregulates SBF2-AS1 to increase RIPK2 by binding miR-196b-5p, which shields against pyroptosis mediated by Caspase-1 to promote its proliferation. These results uncover a novel mechanism of pathogen–host interaction and provide insights for developing new therapeutic strategies against C. trachomatis infection.

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Full text

1. Introduction

C. trachomatis is a Gram-negative obligate intracellular pathogen that can infect the eyes and the reproductive tract, which may even cause severe consequences such as blindness or infertility. With approximately 131 million new cases annually, C. trachomatis genital infection is one of the most prevalent sexually transmitted infections globally [1]. C. trachomatis possesses a unique biphasic developmental cycle, alternating between the infectious elementary body (EB) and the replicative reticulate body (RB). Following entry into the host cell, the EB differentiates into the RB and begins replicating within a membrane-bound compartment known as the inclusion, which facilitates the pathogen’s evading immune recognition and clearance [2].

Pyroptosis is a special form of programmed cell death that plays a crucial role in host defense against microbial infections. Its hallmarks include cell swelling, plasma membrane rupture, and the efflux of pro-inflammatory cytokines [3,4]. During the canonical pyroptosis pathway, inflammatory caspase-1 is activated [5]. Upon activation, it cleaves Gasdermin D (GSDMD), generating the membrane pore-forming N-terminal fragment (GSDMD-N) [6]. This fragment forms pores in the cell membrane [7,8], causing cell swelling and lysis. Furthermore, the host cell can inhibit the growth of C. trachomatis by activating GSDMD-mediated pyroptosis [9].

Many intracellular pathogens have evolved complex strategies to counteract pyroptosis. For example, the effector protein SopB of Salmonella mediates Akt phosphorylation, thereby delaying the activation of caspase-1 and slowing down the pyroptosis process [10]. The phosphatase PtpB secreted by Mycobacterium tuberculosis (M. tuberculosis) can bind to host monoubiquitin and disrupt the membrane localization of GSDMD through dephosphorylation, which blocks the pyroptosis process [11]. C. trachomatis has evolved a unique lipopolysaccharide that does not trigger pyroptosis, enabling it to evade the host’s innate immune response [12]. Could C. trachomatis be employing additional strategies to resist pyroptosis?

LncRNAs are non-protein-coding RNA molecules longer than 200 nucleotides. They play crucial regulatory roles in cellular physiology and pathology, and their functions during pathogen infection are increasingly being recognized [13,14]. Notably, they also play important roles in regulating pyroptosis [15,16]. For example, M. tuberculosis effector protein EST12 promotes pyroptosis through the negative regulation of Lnc-EST12 [17], and lncRNA-Gm17586 inhibits Salmonella typhimurium-mediated pyroptosis by promoting the binding of NLRP3 and Tnip1 [18].

Using high-throughput lncRNA microarray screening, we established that C. trachomatis may reprogram the lncRNA of the host cell and thus its responses to infection [19]. From the microarray dataset (GSE165628), we found that SBF2-AS1 is significantly upregulated in C. trachomatis-infected cells. It is usually related to normal cell proliferation, apoptosis, and migration [20,21,22]. Our study aims to explore the molecular mechanisms by which C. trachomatis regulates host pyroptosis through modulation of SBF2-AS1. The outcomes of this work may lead to new treatments for this widespread disease.

2. Materials and Methods

2.1. Cell Culture and C. trachomatis Infection

HeLa 229 cells were propagated in Dulbecco’s Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum (FBS, Evergreen) at 37 °C under 5% CO_2_. At approximately 70% confluency, cells were inoculated with C. trachomatis serovar E suspended in DMEM (MOI = 1). Following a 30-min incubation at 37 °C, plates were centrifuged (300× g, 45 min, 37 °C) to facilitate infection. Finally, the supernatant was replaced with fresh medium containing 10 µg/mL gentamycin (Merck) under the same culture conditions.

2.2. Reverse Transcription Quantitative Real-Time PCR (RT-qPCR)

Following RNA isolation, cDNA was synthesized using the Vazyme kit. Expression levels were determined by qPCR. SBF2-AS1 was quantified with ChamQ Universal SYBR Mix (Vazyme), with 18S rRNA as the internal control. miR-196b-5p was quantified with the miRNA Universal SYBR qPCR Master Mix (Vazyme), with the U6 snRNA as the endogenous control. All qPCR reactions were conducted in three independent replicates on a LightCycler 96 system (Roche) under a three-step amplification protocol for 40 cycles. Calculations were based on the 2^−ΔΔCt^ method, and the primer sequences are listed in Table 1.

2.3. RNA Interference

Small-interfering RNA (siRNAs) targeting SBF2-AS1 (siSBF2-AS1), miR-196b-5p mimics, and non-targeting scrambled control (scRNA) were synthesized by Sangon Biotech. Following the manufacturer’s protocol, cells were transfected using Lipofectamine 3000 (Invitrogen) upon reaching approximately 50% confluence. The harvested cells were analyzed by RT-qPCR and Western blot to determine transfection efficiency and functional effects after 24 h post-transfection. The siRNA sequence targeting SBF2-AS1 was GCTCCATCAATGCTAGTAT, while the sequence for miR-196b-5p mimics was UAGGUAGUUUCCUGUUGUUGGG.

2.4. Immunofluorescence Assay (IFA)

Cells were fixed in 4% paraformaldehyde for 15 min, rinsed three times with phosphate-buffered saline (PBS), and incubated with 0.25% Triton X-100 for 10 min at room temperature. After washing, cells were blocked using 10% FBS in DMEM medium at 37 °C for 1 h and then incubated with the primary antibody (rabbit anti-GSDMD-N, Abcam-ab215203, 1:200) overnight at 4 °C. Subsequently, the cells were incubated with CoraLite488-conjugated secondary antibody (Proteintech, 1:200) in the dark at 37 °C for 90 min and the nuclei were stained with DAPI (Beyotime). Finally, coverslips were mounted, and images were acquired from a fluorescence microscope (Nikon ECLIPSE Ts2R, Japan). Image analysis was performed using ImageJ 1.53k.

2.5. Bioinformatics Analysis

Potential targets of SBF2-AS1, miR-196b-5p, and RIPK2 were predicted using the miRDB (https://mirdb.org/, accessed on 15 July 2024), ENCORI (https://rnasysu.com/encori/index.php, accessed on 15 July 2024), LncBase (https://diana.e-ce.uth.gr/lncbasev3, accessed on 15 July 2024) databases. Furthermore, we employed miRWalk (http://mirwalk.umm.uni-heidelberg.de/, accessed on 15 July 2024) and ENCORI databases to predict the binding sites where miR-196b-5p binds to RIPK2 and SBF2-AS1.

2.6. Dual-Luciferase Reporter Gene Assay

Based on the predicted interaction sites of miR-196b-5p with both SBF2-AS1 and RIPK2 (via ENCORI and miRWalk), the mutant (MUT) and wild-type (WT) luciferase reporter plasmids were constructed in the psiCHECK-2 vector (Sangon Biotech). The mutant sequences were shown in Table S1. Briefly, the PCR products of RIPK2 and SBF-AS1 were digested with NotI and XhoI and inserted into the vector, respectively. The recombinant plasmids (SBF2-AS1 WT, SBF2-AS1MUT, RIPK2 WT, RIPK2 MUT) were transformed into Escherichia coli competent cells for amplification. Subsequently, these purified plasmids were co-transfected with miR-196b-5p mimics into HeLa cells using Lipofectamine 3000 (Invitrogen) for 48 h. Following the manufacturer’s protocol, the interactions were assessed by a dual-luciferase reporter assay kit (Promega).

2.7. RNA Fluorescent In Situ Hybridization (RNA-FISH)

SBF2-AS1 probe (5′-AAGAACACAACATACTAGCATTGATGGAGCATTG-3′) was synthesized by BersinBio. The 5′ end of the probe was labeled with a Cy3 fluorescent dye. Following fixation and permeabilization, cells were then incubated with the probe in hybridization buffer. After three PBS washes, DAPI (Beyotime) was used for nuclear staining. Fluorescence signals were finally detected using an inverted fluorescence microscope (ECLIPSE Ts2R).

2.8. Western Blot Analysis and Co-IP

Cell lysis was performed on ice for 30 min with 100 μL of RIPA buffer (Solarbio) supplemented with 1 mM PMSF (Solarbio). The lysate was then harvested by scraping and transferred to microcentrifuge tubes. The samples were centrifuged (12,000× g, 10 min, 4 °C), after which the supernatant was collected, and the protein concentration was quantified by the BCA method (Beyotime). Following mixing with loading buffer, protein samples were separated by SDS-PAGE and transferred onto a 0.22 μm PVDF membrane (Millipore). After blocking with 5% non-fat milk, the membrane incubated with the primary antibody (anti-GSDMD, anti-RIPK2, Wanleibio, anti-GSDMD-N, abcam-ab215203, anti-NLRP3, anti-Caspase-1(p10), anti-Caspase-1, Huabio, anti-β-actin, zenbio) overnight at 4 °C. Membranes were washed, incubated with appropriate HRP-conjugated secondary antibodies (Proteintech). Finally, the ECL reagent (Sangon) was applied to the PVDF membrane and incubated in the dark. Images were captured using the G: BoxChemi X × X9 (Syngene). Band density was quantified using Quantity One 25.0 software (BioRad). Protein lysates were co-immunoprecipitated using a commercial Co-IP Kit (MCE) following the manufacturer’s protocols. The immunoprecipitated proteins were assessed by Western blot.

2.9. CCK-8 Assay

HeLa cells were seeded at 2 × 10^3^ cells per well in 96-well plates and cultured for 24 h at 37 °C under 5% CO_2_. Following transfection with siSBF2-AS1, miR-196b-5p mimics, siRIPK2, or their respective controls, HeLa cells were infected with C. trachomatis for another 24 h. After two PBS washes, a mixture of 90 µL medium and 10 µL CCK-8 (Solarbio) was added to each well at 37 °C for 2 h in the dark. The absorbance at 450 nm was recorded using a microplate reader (TECAN Infinite^®^ F50 Plus, Switzerland).

2.10. Statistical Analysis

All statistical analyses and visualization were performed with GraphPad Prism 9. All data were represented as $[eqn]$ ± SD. Group differences were assessed using the Student’s t-test. p < 0.05 was defined as statistically significant.

3. Results

3.1. SBF2-AS1 Participates in Pyroptosis During C. trachomatis Infection

To explore the changes of SBF2-AS1 during the C. trachomatis replication phase, we extracted total RNA at 24 h post-infection from HeLa cells and analyzed the expression levels of SBF2-AS1 by RT-qPCR. Compared to the uninfected group, SBF2-AS1 was upregulated approximately 2-fold (p < 0.001) (Figure 1A).

In order to investigate the relationship between SBF2-AS1 and pyroptosis, we transfected Hela cells with siSBF2-AS1 and control scRNA, respectively. These transfected cells were then either left uninfected or infected with C. trachomatis. After 24 h post-infection, total RNA was isolated from the cells. Compared with the scRNA group, RT-qPCR results showed that SBF2-AS1 was significantly downregulated in the siSBF2-AS1-treated group. The knockdown efficiency of the control group was 81.4% (Figure 1B, p < 0.001), while the C. trachomatis-infected group was 47.9% (Figure 1C, p < 0.05).

After transfecting HeLa cells with siSBF2-AS1 and infecting them with C. trachomatis for 24 h, total protein was extracted. Western blotting analysis showed that the expression of pyroptosis-related proteins GSDMD-N, NLRP3, and Caspase-1 (p10) was significantly upregulated in the siSBF2-AS1 group (Figure 1D–H, p < 0.05). The CCK-8 assay confirmed that cell viability in the siSBF2-AS1 group decreased by 47.25% compared to the control group (Figure 1I, p < 0.001). Fluorescence microscopy showed that GSDMD-N accumulated on the cell membrane of HeLa cells in the siSBF2-AS1 group (Figure 1J). Therefore, SBF2-AS1 is critical for suppression of pyroptosis during C. trachomatis infection.

3.2. SBF2-AS1 Functions as a Sponge for miR-196b-5p in C. trachomatis Infection

To elucidate how SBF2-AS1 participates in pyroptosis, we predicted the location of SBF2-AS1 was in the cytoplasm (Figure 2A) by the AnnoLnc2 (http://annolnc.gao-lab.org/, accessed on 15 July 2024), which was confirmed by RNA-FISH experiments (Figure 2B).

Since SBF2-AS1 was located in the cytoplasm, we sought to identify the potential miRNAs that interacted with it. 121 candidate miRNAs interacting with SBF2-AS1 were screened out from the three databases, such as miRDB (https://mirdb.org/, accessed on 15 July 2024), ENCORI (https://rnasysu.com/encori/index.php, accessed on 15 July 2024), and LncBase (https://diana.e-ce.uth.gr/lncbasev3, accessed on 15 July 2024). Two very similar miRNAs, miR-196a-5p and miR-196b-5p, were found in the three databases (Figure 2C). Due to its association with pyroptosis, miR-196b-5p was chosen for further study [23]. The predicted RNA binding sites of miR-196b-5p and SBF2-AS1 are shown in Figure 2D.

Next, mutant (MUT) and Wild-type (WT) reporter vectors were constructed to validate the interaction sites between miR-196b-5p and SBF2-AS1 via a dual-luciferase reporter assay. The luciferase activity in the SBF2-AS1 WT group was significantly suppressed by miR-196b-5p (Figure 2E, p < 0.05). Moreover, after interfering with SBF2-AS1, the expression of miR-196b-5p significantly increased (Figure 2F, p < 0.05).

3.3. miR-196b-5p Is Involved in Pyroptosis During C. trachomatis INFECTION

As a downstream of SBF2-AS1, can miR-196b-5p also regulate host pyroptosis? After transfecting HeLa cells with miR-196b-5p mimics and infecting them with C. trachomatis for 24 h, total protein was extracted. The results showed that the expression of pyroptosis-related proteins GSDMD-N, NLRP3, and Caspase-1 (p10) was markedly increased in the miR-196b-5p mimics group (Figure 3A–E, p < 0.05). We also found that the cell viability in the miR-196b-5p mimics group decreased by 40.53% (Figure 3F), and GSDMD-N aggregated at the cytoplasmic membrane of HeLa cells (Figure 3G, p < 0.001). These findings showed that miR-196b-5p can regulate the pyroptosis of the host cell.

3.4. SBF2-AS1 Upregulates RIPK2 by Sponging miR-196b-5p During C. trachomatis Infection

Based on the prediction of miRWalk (http://mirwalk.umm.uni-heidelberg.de/, accessed on 15 July 2024) and differentially upregulated mRNAs identified in our previous C. trachomatis infection microarray study [19], we selected RIPK2 for further analysis (Figure 4A). The predicted RNA binding sites of miR-196b-5p and RIPK2 are shown in Figure 4B.

The results demonstrated that miR-196b-5p significantly reduced the luciferase activity of wild-type RIPK2 (Figure 4C, p < 0.05), which verified that RIPK2 was the target of miR-196b-5p. In addition, transfection with miR-196b-5p mimics significantly downregulated the expression of RIPK2 (Figure 4D, p < 0.05). The expression level of miR-196b-5p was detected by RT-qPCR (Figure 4E, p < 0.05). Conversely, interfering SBF2-AS1 can inhibit the expression of RIPK2 (Figure 4F, p < 0.05), suggesting that SBF2-AS1 upregulates RIPK2 by sponging miR-196b-5p. Subsequently, RIPK2 was significantly upregulated during C. trachomatis infection (Figure 4G, p < 0.05). These findings revealed that C. trachomatis enhanced the level of RIPK2 via the SBF2-AS1/ miR-196b-5p axis.

3.5. SBF2-AS1 Resists Pyroptosis to Promote C. trachomatis Growth by Regulating the RIPK2

Given that miR-196b-5p targeted RIPK2, how was RIPK2 involved in the regulation of pyroptosis? The knocking down of RIPK2 could upregulate the expression of these pyroptosis-related proteins (Figure 5A–E, p < 0.05). Meanwhile, GSDMD-N aggregated on the cytoplasmic membrane of HeLa cells (Figure 5F). The CCK-8 assay also confirmed that cell viability decreased by 46.91% in the RIPK2 knockdown group (Figure 5G, p < 0.001). To explore how RIPK2 exerts its effect on pyroptosis, we used the online database BioGRID (https://thebiogrid.org/, accessed on 18 August 2024) to predict the potential interaction between RIPK2 and Caspase-1. The interaction between RIPK2 and Caspase 1 was verified by Co-IP assays (Figure 5K).

After transfection with siSBF2-AS1, miR-196b-5p mimics, siRIPK2, and their respective controls, HeLa cells were infected with C. trachomatis for another 24 h. The results showed that the infection rate of C. trachomatis was reduced in the three treatment groups compared to the control group. The reduction rates were 55.00% (Figure 5H, p < 0.001), 44.47% (Figure 5I, p < 0.001), and 34.83% (Figure 5J, p < 0.01), respectively. The results indicate that C. trachomatis may protect itself against Caspase-1-mediated pyroptosis through the SBF2-AS1/miR-196b-5p/RIPK2 axis and promote its own growth and development further.

4. Discussion

In this study, we revealed a novel mechanism employed by C. trachomatis to suppress pyroptosis of host cells. It was shown that SBF2-AS1 was significantly upregulated at 24 h postinfection in HeLa cells and was localized to the cytoplasm. This suggested that SBF2-AS1 may regulate host cell responses in the cytoplasm by modulating mRNA stability or miRNA function. Other researchers have reported similar findings [24]. For example, Acinetobacter baumannii (A. baumannii) upregulates lncRNA-GAS5, leading to STX17 degradation, which enhances replication of A. baumannii [25]. LncRNA-CFTBS facilitates M. tuberculosis intracellular survival by binding miR-515-5p/miR-519e-5p to regulate SAT1 expression [26]. SBF2-AS1 serves as a competitive endogenous RNA (ceRNA) in the cytoplasm and further regulates gene expression by targeting miRNAs [27]. In this study, silencing of SBF2-AS1 significantly increased the expression of pyroptosis-related proteins GSDMD-N, NLRP3, and Caspase-1 (p10), and decreased cell viability, indicating that SBF2-AS1 may play a crucial role in regulating pyroptosis.

To elucidate the molecular roles of SBF2-AS1, we employed bioinformatics analyses and dual-luciferase reporter assays to confirm that SBF2-AS1 may function as a molecular sponge for miR-196b-5p. miR-196b-5p is involved in the occurrence and development of various diseases [28,29,30]. Reduced miR-196b-5p upregulates the expression of MGAT4A, which induces apoptosis and suppresses the tumor [31]. In our study, C. trachomatis infection significantly downregulated miR-196b-5p, silencing SBF2-AS1 partially reversed this effect. This indicates that C. trachomatis may suppress the function of miR-196b-5p by upregulating SBF2-AS1. Overexpression of miR-196b-5p significantly enhanced the expression of pyroptosis-related proteins, suggesting that miR-196b-5p itself has a pro-pyroptotic effect.

Bioinformatics analysis identified RIPK2 as a target of miR-196b-5p, and the dual-luciferase reporter assay confirmed the interaction between miR-196b-5p and RIPK2. RIPK2 is a serine/threonine kinase that plays an important role in innate immunity, especially in the NOD-like receptor (NLR) signaling pathway [32]. During C. trachomatis infection, the expression of RIPK2 was increased. However, silencing SBF2-AS1 and overexpressing miR-196b-5p led to a decrease in RIPK2 expression, suggesting that C. trachomatis upregulated RIPK2 through the SBF2-AS1/miR-196b-5p axis. Knockdown of RIPK2 significantly promoted the occurrence of pyroptosis. Further Co-IP experiments confirmed that RIPK2 directly binds to Caspase-1, inhibiting its cleavage activity, thereby blocking the generation of GSDMD-N and the occurrence of pyroptosis.

Our study reveals that during its critical replication phase, C. trachomatis upregulates the expression of SBF2-AS1 to target miR-196b-5p. This interaction attenuates the inhibitory effect of miR-196b-5p on RIPK2, thereby enhancing the expression of RIPK2. Subsequently, RIPK2 interacts with Caspase-1to block the pyroptosis pathway. This strategic inhibition of host cell pyroptosis allows C. trachomatis to maintain its intracellular niche, providing favorable conditions for its proliferation (Figure 6). How C. trachomatis upregulates SBF2-AS1 needs further study. However, previous research suggests a potential pathway. Knockdown of LncRNA ZEB1-AS1 downregulates the expression of transcription factor ZEB1 [33], which activates SBF2-AS1 by binding directly to the promoter region [34]. Given that C. trachomatis infection upregulates ZEB1-AS1 [35], this pathogen may enhance the expression of SBF2-AS1 through the ZEB1-AS1/ ZEB1 axis.

In summary, by confirming the role of the SBF2-AS1/miR-196b-5p/RIPK2 axis in C. trachomatis reproduction, this study provides novel insights into host-pathogen interactions and reveals potential therapeutic targets for combating C. trachomatis infections.