Transcriptomic Profiling of TSST-1–Induced Cytotoxicity and Inflammatory Responses in Human Vaginal Epithelial Cells

Transcriptomic Profiling of TSST-1–Induced Cytotoxicity and Inflammatory Responses in Human Vaginal Epithelial Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptomic Profiling of TSST-1–Induced Cytotoxicity and Inflammatory Responses in Human Vaginal Epithelial Cells XUELING LUO, yue yuan, ke chen, changfeng sun, hong yu, Suvash Chandra Ojha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8800664/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Objective Toxic shock syndrome toxin-1 (TSST-1) is a critical superantigen produced by certain staphylococcal strains, closely associated with menstrual toxic shock syndrome and mucosal inflammation. While the systemic superantigenic function of TSST-1 is well-established, its early cytotoxic and inflammatory effects on human vaginal epithelial cells (HVEC) remain poorly characterized. This study, therefore, seeks to investigate the initial temporal dynamics of TSST-1-induced cell viability, inflammatory cytokine responses, and barrier disruption in a model of human vaginal epithelium, along with a detailed analysis of the transcriptome. Methods We evaluated the impact of recombinant TSST-1 on HVEC at different doses and treatment durations. Subsequent exposure assessments included cell viability, apoptosis, and cell cycle distribution via flow cytometry, alongside inflammatory gene expression and transcriptomic profiling. Results TSST-1 could significantly reduce cell viability and proliferation in a dose- and time-dependent manner, accompanied by increased apoptosis and marked G1 phase arrest. In contrast, early exposure did not result in a uniform upregulation of all inflammatory mediators; rather, multiple cytokines exhibited dose-dependent segmented response patterns instead of a linear increase. RNA sequencing analysis of samples treated with 125 or 1000 ng/mL TSST-1 for 48 hours revealed extensive transcriptional reprogramming involving chemokine signaling, cytokine-receptor interactions, epithelial stress-related pathways, and metabolic rewiring. Notably, the number of differentially expressed genes induced by moderate-dose TSST-1 exceeded that of high-dose treatment, while high-dose TSST-1 specifically enriched pathways related to hypoxia and glycolysis. Conclusion These findings demonstrate that TSST-1 can trigger cytotoxicity, apoptosis, cell cycle disruption, and a graded inflammatory response in vaginal epithelial cells, providing a systematic transcriptomic framework for understanding the early mucosal responses induced by TSST-1. TSST-1 human vaginal epithelial cells cytotoxicity apoptosis cell-cycle inflammatory factors RNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Toxic shock syndrome (TSS) is a life-threatening systemic disorder triggered by superantigens (SAgs) secreted by certain Staphylococcus aureus or Streptococcus pyogenes [ 1 , 2 ]. Clinically, TSS typically begins with transient, nonspecific prodromal symptoms and can rapidly progress to hypotension, shock, and multiorgan failure [ 3 ][ 4 ]. Among staphylococcal superantigens, toxic shock syndrome toxin-1 (TSST-1) is the prototypical virulence factor: it binds major histocompatibility complex class II (MHC II) molecules on antigen-presenting cells and crosslinks them with the Vβ domain of T-cell receptors, bypassing conventional antigen processing and driving massive, non-specific T-cell activation with excessive release of TNF-α, IL-1, IL-2, IFN-γ, and other mediators, culminating in a cytokine storm [ 5 – 7 ]. Epidemiological observations in the 1980s, when TSST-1–associated menstrual TSS rose sharply in parallel with high-absorbency tampon use, further pointed to the female reproductive tract as a critical portal of exposure; risk was associated with prolonged single-tampon wear, overnight use, and suboptimal insertion practices [ 8 – 10 ]. In this context, the vaginal epithelium is not only a physical barrier but also an immunologically active interface that produces antimicrobial peptides, transports IgA, secretes chemokines, and engages in hormone-dependent (e.g., estrogen-regulated) crosstalk with local and systemic immunity[ 11 , 12 ]. Clinically, current management strategies focus on rapid control of infection and containment of systemic consequences, including empirical broad-spectrum antibiotics, source control via imaging-guided intervention or surgical debridement when needed, and prompt removal of foreign materials such as tampons, nasal packing, or wound dressings [ 3 , 13 ]. Combination regimens that pair β-lactams with clindamycin are commonly used, as clindamycin suppresses bacterial protein synthesis and reduces superantigen production [ 14 ]. However, no approved therapy directly neutralizes TSST-1, and existing approaches primarily target bacterial load or systemic inflammation rather than the initial mucosal events. This gap underscores the need to refocus attention on the vaginal mucosa as a frontline site where TSST-1 is sensed, where barrier integrity may be compromised, and where early inflammatory programming could shape downstream systemic responses. As part of this frontline, tight junction proteins such as occludin, claudin-1, and ZO-1 are essential for barrier maintenance [ 15 ], while pattern recognition receptors (PRRs) enable epithelial cells to detect microbial and toxin-associated signals and rapidly secrete TNF-α, IL-1β, IL-6, chemokines, and antimicrobial peptides to recruit neutrophils and restrict bacterial dissemination [ 16 ]. Despite extensive work on TSST-1–induced systemic immune dysregulation and cytokine storm, direct evidence delineating its early impact on human vaginal epithelial cells (HVEC) remains limited. Prior in vitro studies have often focused on single endpoints such as isolated cytokine readouts or barrier-related markers without integrating cytotoxicity, proliferative capacity, cell-cycle dynamics, apoptosis, and inflammatory signaling into a unified framework [ 17 – 19 ]. To address this knowledge gap, we employed the HVEC line VK2/E6E7 and exposed it to graded concentrations of recombinant TSST-1 under time-controlled conditions, with the aim of answering three interrelated questions: (i) whether TSST-1 induces measurable cytotoxicity in a dose- and time-dependent manner; (ii) whether the balance between proliferation and programmed cell death shifts, reflected by changes in apoptosis rates and cell-cycle distribution; and (iii) whether epithelial inflammatory programs are activated at both the transcriptional and protein levels. Here, we integrated metabolic and growth assays to evaluate cell viability, employed flow cytometry to analyze apoptosis and cell-cycle status, and quantified representative cytokines at both mRNA and secretory levels, while correlating these phenotypic data with RNA-seq and pathway enrichment analyses to map TSST-1–responsive signaling networks. By linking epithelial-level cytotoxicity, proliferative restraint, and apoptosis with transcriptional remodeling of barrier- and immune-related pathways, this study provides a mucosal perspective that helps connect local epithelial perturbations to the broader systemic dysregulation observed in TSS, thereby offering a basis for earlier detection and targeted intervention strategies. Simultaneously, emerging evidence indicates that anti-virulence compounds targeting the S. aureus SaeRS two-component system can suppress TSST-1 production without exerting strong bactericidal pressure, thereby supporting the feasibility of interventions aimed at modulating toxin regulation rather than solely relying on conventional antibiotics [ 20 ], reinforcing the relevance of dissecting TSST-1’s direct effects on vaginal epithelial cells. 2. Materials and Methods 2.1 Construction of His-tagged Recombinant Plasmid The TSST-1 gene sequence was retrieved from NCBI with the GenBank accession number J02615. The synthetic TSST-1 gene sequence, featuring a deleted stop codon and incorporating BamH1 and XhoI restriction sites at the 5' and 3' ends, respectively, was introduced into the pET28a(+) expression vector (SciProtech, Beijing) as a fusion with a His-tag (H 6 ) and SUMO (small ubiquitin-like modifier). The sequence of the recombinant plasmid (TSST-H6-SUMO-pET28a (+)) was validated using restriction digestion and DNA sequencing to ensure its integrity and the absence of mutations, and it was subsequently transformed into Escherichia coli NiCo21 (DE3) for expression and purification. 2.2 Protein Expression and Verification A single colony of E. coli NiCo21 (DE3) harboring the tsst expression plasmid was inoculated into 10 mL of LB medium and cultured overnight at 37°C with shaking (220 rpm). The culture was diluted 1:100 into 500 mL of fresh LB medium and grown to mid-log phase (OD₆₀₀ ~0.6). Protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) and incubated for 20 h at 37°C. Cells were harvested by centrifugation (9000 rpm, 4°C, 30 min), resuspended in RIPA buffer (Beyotime, P0013B), and lysed on ice for 30 min with gentle intermittent vortexing. Following clarification of the lysate by centrifugation (12 000 rpm, 4°C, 15 min), the resultant supernatants, representing the soluble protein fractions, were analyzed by 12% SDS-PAGE alongside uninduced controls to confirm expression and solubility. Additionally, the identity and tag status of the resulting protein were subsequently verified by Western blotting. Briefly, samples resolved by SDS–PAGE were electrotransferred onto a nitrocellulose membrane, blocked with a protein-free rapid blocking solution (PS108P, EpiZyme) at room temperature, and incubated with a mouse anti-His tag monoclonal antibody (AE003, Abclonal), followed by a goat anti-mouse IgG secondary antibody (Servicebio). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) detection system (Omni-ECL™, EpiZyme) according to the manufacturer’s instructions. 2.3 Protein Purification Filtered supernatants were loaded onto an affinity-based Ni²⁺-NTA gravity column (5-mL HisTrap™ HP) pre-equilibrated with Buffer A (20 mM phosphate buffer, pH 7.4, 0.5 M NaCl, 0.05% Tween-20, 20 mM imidazole). The column was washed with 5–10 column volumes (CV) of Buffer A before being eluted stepwise with 50, 100, 250, and 500 mM imidazole (each 5 CV) from Buffer B (500 mM imidazole). Protein fractions were screened by 12% SDS-PAGE. The 50 mM eluate was treated with ubiquitin-like-specific protease (ULP1, ~ 10 U per 50 µg protein) at 30°C for 2 h to remove the SUMO tag, then reloaded onto the Ni²⁺-NTA column to collect flow-through fractions. The final fractions were pooled for anion-exchange (Q) chromatography following sequential 20 mM and 500 mM imidazole wash/elution cycles. Except for protease digestion at 30°C, all steps were performed at 4°C to prevent degradation. 2.4 His-tag Removal and Post-Cleavage Purification Purified His-tagged rTSST-1 was transferred into cleavage buffer and digested with site-specific protease at the engineered linker to remove the N-terminal His tag. Aliquots were periodically collected, mixed with 2× SDS loading buffer to terminate the reaction, and analyzed by reducing 12% SDS-PAGE. After complete digestion, the sample was buffer-exchanged into phosphate buffer (pH 7.4) containing 20 mM imidazole and 0.5 M NaCl, and was subsequently reloaded into a Ni²⁺-NTA column. The untagged rTSST-1 was collected in the flow-through and low-imidazole wash fractions, while residual His-tagged and any uncleaved proteins were retained on the matrix. All chromatographic steps were conducted at 4°C, and fractions containing the target proteins were verified by SDS-PAGE, yielding homogenous tag-free rTSST-1 for subsequent assays. 2.5 Cell Culture The HVEC (VK2/E6E7) was purchased from iCell. Cells were maintained in serum-free VK2/E6E7 medium (iCell-h551-001b, 500 mL) supplemented with the manufacturer’s recommended growth factors, 1% FBS (SPERIKON SP010010500) and 1% penicillin/streptomycin. This medium formulation was used for both routine culture and subsequent toxin exposure experiments. Cultures were maintained in a humidified incubator at 37°C with 5% CO₂. Upon reaching 80% confluence, cells were passaged using 0.25% trypsin-EDTA (HyClone SH30042.01) for approximately 4 min at 37°C. Trypsinization was halted by adding medium containing 5% FBS or through centrifugation and resuspension in fresh medium. Cells from early passages (P5–P15) were employed for all experiments. The culture medium was replaced 2 to 3 times per week, and cells were passaged at ratios of 1:2 to 1:5 every 4 to 6 days [ 21 ]. 2.6 Cell Viability Assay VK2/E6E7 cells were seeded at 3000 cells/well in 96-well plates and pre-cultured for 24 h. Cells were then treated with rTSST-1 at final concentrations of 0, 60, 125, 250, 500, and 1000 ng/mL for 24, 48, 72, and 96 h. Each condition was performed in five technical replicates across three distinct biological experiments, with wells randomized across plates. Following treatment, 10 µL of CCK-8 reagent (GLPBIO GK10001-30) was added to each well, plates were incubated for 2 h at 37°C, and absorbance was measured at 450 nm. The viability of each treatment group was standardized to the untreated control (0 ng/mL) and is expressed as mean ± standard deviation (SD). 2.7 Cell Proliferation Assay Cell proliferation was assessed using EdU labeling. VK2/E6E7 cells were seeded in 24-well plates and treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 48 h. Pre-warmed 2× EdU working solution was added to a final concentration of 10 µM and incubated for 4 h. Subsequently, cells were fixed with 4% paraformaldehyde (15 min), permeabilized with 0.3% Triton X-100/PBS (15 min), and stained using Azide-594 click reaction (30 min) in the dark. Nuclei were counterstained with Hoechst 33342 for 10 min. Fluorescence images were captured (Ex/Em 590/615 nm) and the proliferation rate (EdU⁺/Hoechst⁺ ratio) was assessed from a minimum of five fields per well across two independent experiments. 2.8 Apoptosis Detection Assay Apoptosis was quantified using the BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit (556547). VK2/E6E7 cells in 6-well plates were treated with 0, 250, 500, or 1000 ng/mL TSST-1 for 48 h. Following two washes with cold PBS, 1×10⁵ cells were resuspended in 100 µL of 1× binding buffer, stained with 5 µL of FITC Annexin V and 5 µL of PI, and incubated for 15 min at room temperature in the dark. After adding 400 µL of binding buffer, samples were analyzed within 1 h on a BD FACSAria cytometer. Triplicate biological replicates were processed, and populations (live, early apoptotic, and late apoptotic/necrotic) were quantified using FlowJo. 2.9 Live/dead Cell Staining Live/dead ratios were visualized by Calcein AM/PI staining (Beyotime, C2015M). VK2/E6E7 cells in 24-well plates were treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 48 h, washed once with PBS, and incubated with 250 µL of Calcein AM/PI working solution at 37°C for 30 min in the dark. Images were captured on an Olympus IX73 microscope using green (Calcein AM, Ex/Em 494/517 nm) and red (PI, Ex/Em 535/617 nm) channels. Two biological repeats were performed, analyzing ≥ 5 random fields per well for Calcein⁺ and PI⁺ cell percentages. 2.10 Cell Cycle Analysis VK2/E6E7 cells were treated with TSST-1 (0-1000 ng/mL) for 72 h, subsequently harvested, and fixed overnight in 75% ethanol at − 20°C. Following washing, cells were stained with 0.5 mL of PI/RNase solution (BD Pharmingen) for 15 min at room temperature in the dark, then stored at 4°C and analyzed within 1 h using a BD FACSAria cytometer (BD Biosciences, instrument ID TY2013001909). Modfit was used to compute cell-cycle distributions (G₀/G₁, S, G₂/M) across three independent biological replicates. 2.11 Quantitative Real-Time PCR Total RNA was extracted from cells following a 24 h treatment with TSST-1 (0, 500, 1000 ng/mL) or 1000 ng/mL LPS (positive control) using a commercial kit (TIANGEN DP419). The purity and integrity of RNA were assessed using NanoDrop and agarose gel electrophoresis, respectively. One microgram of RNA was reverse-transcribed using SweScript All-in-One RT SuperMix (Servicebio G3337)- Quantitative PCR (qPCR) was performed in triplicate technical wells on a QuantStudio 3 system employing 2× SYBR Green Master Mix (High ROX, Servicebio G3322-05) under the following cycling program: 95°C for 30 s; 40 cycles of 95°C for 15 s, 60°C for 10 s, 72°C for 30 s; followed by melting curve analysis. The targets included IL-1β, IL-6, IL-8, CCL3, TNF, and CXCL1, with β-actin serving as an internal control. Relative expression was determined using the 2 − ΔΔCt technique. 2.12 RNA-seq VK2/E6E7 cells were treated with 125 ng/mL (low) or 1000 ng/mL (high) of rTSST-1 for 48 h, while untreated cells served as negative controls (NC), and were processed in triplicate. Total RNA was extracted using TRIzol and subsequently submitted to Novogene for library construction and sequencing. Raw FASTQ reads were cleaned with SeqPrep and Sickle, then aligned to the reference genome using Tophat2. Gene/transcript expression was quantified with HTSeq, and differential expression was analyzed by DESeq with a threshold of p < 0.05. Differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify pathways modulated by TSST-1. 2.13 Lactate/Pyruvate Ratio Assay The lactate-to-pyruvate (L/P) ratio was quantified in triplicate using a commercial enzymatic assay kit (Servicebio), following the manufacturer's protocols. Briefly, supernatants from VK2/E6E7 cells treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 24 or 48 h were collected. To quantify lactate, 50 µL of each sample was combined with 44 µL of lactate assay buffer, 2 µL of Amplex Red, 2 µL of Enzyme Solution A, and 2 µL of Enzyme Solution B to prepare a 100 µL reaction mixture. To test pyruvate, 72 µL of pyruvate assay buffer, 2 µL of Amplex Red, 2 µL of Enzyme Solution A, 2 µL of Enzyme Solution B, and 2 µL of cofactor were combined to make an 80 µL working solution. All reactions were incubated in the dark for 30 minutes at room temperature, and absorbance was measured at 560 nm. The final L/P ratios were derived using standard curves and normalized to the control group (0 ng/mL), with data expressed as mean ± SD. 2.14 Statistical Analysis All experiments were independently performed at least three times, and the data are presented as the mean ± SD. The statistical significance between two groups was evaluated using a two-tailed Student’s t-test, whereas comparisons across multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s post hoc test, as applicable. For data that did not satisfy the assumptions of normality or homogeneity of variance, corresponding nonparametric tests were applied. Statistical analyses were performed using GraphPad Prism 9.0 software, and P < 0.05 was considered statistically significant. Differentially expressed genes (DEGs) in transcriptomic data were identified using the DESeq2 package with thresholds of |log₂FC| ≥ 1 and adjusted q < 0.05 (Benjamini–Hochberg correction). 3. Results 3.1 Soluble Expression, Verification and Purification of rTSST-1 (A) SDS-PAGE profile showing total protein before and after induction, and in crude lysates prior to purification. Lane M: molecular weight markers (kDa); Lane 1: total proteins from uninduced cells; Lane 2: total proteins from IPTG-induced cells; Lane 3: soluble fraction of lysed cells prior to chromatography. (B) SDS-PAGE analysis of Ni-NTA affinity-purified fractions shows a prominent band at the expected molecular weight, indicating effective enrichment of the His-fusion protein. (C) Western blot analysis using an anti-His antibody specifically detected a single immunoreactive band at the expected molecular weight in the induced (Lane 2) and Ni-NTA–purified sample (Lane 3), whereas no signal was observed in the uninduced control (Lane 1). Lane M represents molecular weight markers in kDa. (D) SDS-PAGE after removal of the fusion/His tag exhibited a sharp single band with minimal background (Lane 1), thereby confirming successful cleavage. For successful application of TSST-1 protein in cellular assays, the r TSST-1 gene was cloned into the pET-28a(+) vector to generate an N-terminal His-tagged construct. This construct was expressed in E. coli under IPTG induction and purified using Ni-NTA affinity chromatography, followed by site-specific protease cleavage and re-chromatography to remove the His tag. The expression, solubility, purification efficiency, and tag removal of rTSST-1 were then verified, and each stage was examined using SDS-PAGE. No distinct band was observed at the target molecular weight in uninduced samples, whereas a prominent band appeared at approximately 39 kDa after IPTG induction and remained detectable in the soluble fraction (Fig. 1 A), indicating predominantly soluble expression. Ni-NTA affinity purification yielded a single ~ 39 kDa band with minimal impurities (Fig. 1 B). For further validation, purified rTSST-1 was analyzed by Western blotting using an anti-His antibody (Fig. 1 C). A clear immunoreactive band was detected at approximately 39 kDa in lanes 2 and 3, whereas no specific signal was observed in lane 1, confirming successful expression and purification of His-tagged rTSST-1 with high specificity. Following tag removal, the major band shifted to ~ 26 kDa (Fig. 1 D), which corresponds to the expected theoretical molecular weight of tag-free TSST-1, confirming complete cleavage and intact protein integrity. These results demonstrate that recombinant TSST-1 was successfully expressed in soluble form, purified to high homogeneity, and effectively de-tagged, meeting the requirements for subsequent cellular experiments. 3.2 TSST-1 Inhibits the Viability and Proliferation of Vaginal Epithelial Cells in a Time- and Dose-Dependent Manner (A) CCK-8 time-course curves of cell viability (0–96 h) after treatment with rTSST-1 (0–1000 ng/mL). (B) Bar charts show cell viability at 24, 48, 72, and 96 h, expressed as a percentage of the untreated control (mean ± SD, n = 3). (C) EdU staining images show proliferating nuclei (EdU⁺, red) and total nuclei (Hoechst, blue). (D) Quantification of EdU-positive areas at different concentrations. (E) Phase-contrast micrographs show cell morphological changes in response to increasing TSST-1 concentrations and prolonged exposure. To further assess the effects of TSST-1 on VK2/E6E7 cells, viability (CCK-8), proliferation (EdU), and morphological alterations were analyzed over a period of 96 h. Compared to the control, the CCK-8 curves revealed a gradual decline in viability with increasing toxin concentration and prolonged exposure: with the exception of the highest dose (1000 ng/mL), all treatment groups showed diminished increases over time, remaining consistently below control levels, with widening disparities observed between 24 and 96 h. The 1000 ng/mL curve consistently exhibited the lowest and was virtually flat throughout (Fig. 2 A). Bar-plot analysis showed that at 24 h, only the high-dose group exhibited a noticeable drop; at 48 h, viability suppression became significant from mid to high doses, and by 72–96 h, all doses displayed distinct and graded declines (p < 0.05; Fig. 2 B). EdU staining confirmed this trend: red EdU⁺ nuclei decreased progressively with higher toxin levels, and merged images revealed a marked reduction of the red–blue overlap region. The quantitative analysis of EdU⁺ areas showed consistent, statistically significant dose-dependent reductions (p < 0.05; Fig. 2 D, 2 E). Under phase-contrast microscopy, cells transitioned from densely adherent and dispersed morphologies to rounder, shrunken forms with widened intercellular spaces and partial detachment. These morphological changes intensified from 24 h to 96 h, most prominently in the high-dose group (Fig. 2 C). Collectively, these results demonstrate that TSST-1 exerts both time- and concentration-dependent inhibitory effects on epithelial cell viability and proliferation, along with progressive morphological deterioration. 3.3 TSST-1 Induces Enhanced Apoptosis and Disrupts the Cell Cycle (A) Flow cytometric analysis using Annexin V-FITC/PI double staining, showing changes in early and late apoptotic cell populations following treatment with increasing concentrations of TSST-1. The right panel presents quantitative apoptosis rates (mean ± SD, n = 3). (B) Calcein-AM/PI live–dead cell staining images, where green fluorescence indicates live cells and red fluorescence indicates dead cells, with corresponding quantitative analysis on the right. (C) Cell-cycle distribution histograms after exposure to different TSST-1 concentrations for 24, 48, and 72 h. With increasing toxin concentrations, the proportion of cells in the G₁ phase increased, while the S-phase fraction decreased, suggesting cell-cycle arrest. Collectively, these results demonstrate that TSST-1 markedly promotes apoptosis and induces G₁-phase blockade, thereby suppressing epithelial cell proliferation. To further substantiate the cellular effects of TSST-1, apoptosis (Annexin V/PI), live/dead cell ratios (Calcein-AM/PI), and cell-cycle distribution were analyzed after 24 h of treatment. In comparison to the control (Fig. 3 A, 3 B), early and late apoptotic populations increased notably at 250, 500, and 1000 ng/mL, with apoptosis rates rising dose-dependently and achieving statistical significance (p < 0.05). Calcein-AM/PI staining revealed a gradual reduction of viable (green) cells and a corresponding rise in dead (red, PI-positive) cells with increasing toxin concentration, consistent with quantitative analysis showing a significant dose-dependent decline in viability (p < 0.05; Fig. 3 C, 3 D). Furthermore, cell-cycle analysis indicated that increased TSST-1 exposure boosted the proportion of cells in the G 1 phase while reducing those in the S phase, with both changes strongly correlated with concentration (p < 0.05; Fig. 3 E), implying cell-cycle arrest. These results collectively demonstrate that TSST-1 markedly promotes apoptosis and induces G₁-phase arrest, thereby suppressing epithelial cell proliferation. 3.4 Dose-dependent Activation of Epithelial Proinflammatory Responses by TSST-1 Quantitative RT-qPCR measured the mRNA expression levels of inflammation-related genes: (A) IL-1β, (B) IL-6, (C) IL-8, (D) MIP-α, (E) TNF-α, and (F) CXCL1 following treatment with TSST-1 at concentrations of 0, 500, and 1000 ng/mL, with the LPS group serving as a positive control. Results are expressed as fold change relative to the control group (mean ± SD, n = 3). TSST-1 significantly upregulated the transcription levels of multiple proinflammatory genes, indicating its ability to activate the inflammatory response pathways in vaginal epithelial cells. To evaluate the proinflammatory response induced by TSST-1, we performed qPCR validation and quantitative analysis of key cytokine and chemokine genes involved in inflammatory signalling pathways, including IL-1β, IL-6, IL-8, TNF, CXCL1, and CCL3 (MIP-α). Figure 4 illustrates the y-axis denoting fold change, with groups consisting of 0, 500, and 1000 ng/mL TSST-1, as well as LPS. Compared to the untreated control, IL-1β and IL-6 increased significantly at 500 ng/mL and remained elevated at 1000 ng/mL. However, there was a “low-dose higher than high-dose” trend, demonstrating a non-linear pattern. In contrast, IL-8, MIP-α, TNF-α, and CXCL1 showed little or no change at 500 ng/mL but were significantly upregulated at 1000 ng/mL, with overall expression levels comparable to or exceeding those of the LPS positive control. These results indicate that TSST-1 exhibits a graded response among different inflammatory factors: IL-1β and IL-6 reach their maximum at lower doses, whereas IL-8, MIP-α, TNF-α, and CXCL1 increase with higher concentrations. 3.5 Transcriptomic Profiling Reveals Core Response Pathways and Dose-dependent Regulation (A) Volcano plots showing differentially expressed genes (DEGs) in low-dose (125 ng/mL) and high-dose (1000 ng/mL) TSST-1 treatments relative to control (NC); red indicates upregulation, while blue denotes downregulation. (B) A Venn diagram and bar chart displaying the overlap and unique DEGs among the two treatment groups and the control group. (C) Principal component analysis (PCA) showing obvious distinction among the three groups, indicating pronounced transcriptional divergence following TSST-1 treatment. (D) GO enrichment bubble plot illustrating significantly altered biological processes, including chemotaxis, antimicrobial defense, and receptor–ligand modulation. The transcriptomic effects of TSST-1 were meticulously investigated by comparing RNA-seq profiles of the control (NC), low-dose (TSST low), and high-dose (TSST high) groups. Relative to the control, the TSST-low group exhibited 898 upregulated and 581 downregulated genes, whereas the TSST-high group showed 726 upregulated and 378 downregulated genes, indicating that TSST-1 elicited a broader transcriptional response at a lower toxin concentration (Fig. 5 A). The Venn diagram analysis demonstrated a substantial overlap of differentially expressed genes between the two treatment groups, while each condition retained distinct DEG subsets, indicating the coexistence of conserved core transcriptional responses alongside dose-dependent variations (Fig. 5 B). Principal component analysis revealed distinct spatial separation among NC, TSST-low, and TSST-high samples, with tight clustering of biological replicates within each group, indicating pronounced transcriptional divergence following TSST-1 stimulation and supporting the reproducibility and robustness of the sequencing data (Fig. 5 C). Functional enrichment analysis employing Gene Ontology showed that both TSST-low and TSST-high groups were predominantly enriched in immune-related biological processes, including receptor–ligand regulation, antiviral defense, and chemotactic signaling. In contrast, the high-dose condition further enriched stress-associated terms such as hypoxia response, cytoskeletal organization, and ion channel–related activities, suggesting a shift from immune activation to cellular stress and functional remodeling at elevated TSST-1 concentrations (Fig. 5 D). (A) KEGG enrichment bubble plot: the low-dose group was mainly enriched in “cytokine–cytokine receptor interaction,” “chemokine signaling,” and classical immune pathways, including IL-17, TLR, and NOD-like receptor signaling; the high-dose group exhibited additional involvement in PI3K-Akt, VEGF, HIF-1, glycolysis/gluconeogenesis, arginine–proline metabolism, and PPAR pathways. (B) Bar charts illustrating the quantitative expression (log₁₀(FPKM + 1)) of genes in the cytokine–cytokine receptor interaction pathway, alongside corresponding qPCR results wherever applicable. A majority of chemokine and interleukin genes were downregulated in the treated groups, while a few receptors or ligands showed slight upregulation. (C) Protein–protein interaction (PPI) network illustrating key regulatory nodes and modular rearrangements under low- and high-dose conditions, highlighting a dose-dependent network reorganization centered on the cytokine–chemokine axis. KEGG analysis was consistent with these observations. The low-dose group was enriched in “cytokine–cytokine receptor interaction,” “chemokine signaling,” and classical immune pathways, including IL-17, TLR, and NOD-like receptor signaling, whereas the high-dose group extended to PI3K-Akt, VEGF, HIF-1, glycolysis/gluconeogenesis, arginine–proline metabolism, and PPAR pathways (Fig. 6 A). To visualize changes in immune- and chemokine-related genes more intuitively, a bar chart of log 10 (FPKM + 1) values was generated. Most chemokine and interleukin genes (e.g., CXCL2 , CXCL3 , CXCL10 , IL24 , IL1RN ) showed reduced expression compared to the control, while a few receptors or ligands were slightly upregulated, suggesting potential feedback regulation within the overall inflammatory activation (Fig. 6 B). Simultaneously, the corresponding PPI network showed that key nodes clustered around the cytokine–chemokine axis, with the strength of network connections and module organization rearranged in a dose-dependent manner (Fig. 6 C). (A) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in the TSST-high and TSST-low groups. High-dose TSST-1 treatment was enriched in pathways related to interferon signaling, GPCR signaling, and potassium (K⁺) channel activity, while low-dose treatment primarily enriched pathways related to ion transport and neuronal processes. (B) Heatmaps of representative DEGs. The TSST-high group showed marked upregulation of IFIT1/2/3, OASL, HK2, CA9, and KRT16, whereas the TSST-low group specifically upregulated CXCL3, PPP1R14A, NECTIN4, TXNIP, and G0S2. (C) The lactate/pyruvate ratio in culture supernatants elevated with increasing TSST-1 concentration at both 24 h and 48 h, exhibiting a notable peak at 1000 ng/mL. KEGG pathway analysis revealed that cells treated with high-dose TSST-1 showed significant enrichment of innate immune-related pathways, particularly interferon signaling, along with activation of GPCR signaling and potassium (K⁺) channel regulation pathways(Fig. 7 A). In contrast, low-dose TSST-1 primarily affected ion transport, glutamate neurotransmitter release, and other neuronal signaling–related pathways. Consistent with these pathway-level differences, we also compared the 20 genes exhibiting the most significant differential expression following TSST-1 treatment. Heatmap analysis of representative differentially expressed genes revealed that the transcriptional responses to high-dose TSST-1 and low-dose TSST-1 largely overlapped in glycolysis-related genes yet affected a distinct set of genes involved in cellular signalling and metabolic regulation (NDRG1, ENO2, HK2, LOX, SLC2A3, ARG2, CA9, ADSSL1, STC1). This suggests that the TSST-1's effects on epithelial cells are closely linked to glycolysis(Fig. 7 B). Consequently, we measured lactate and pyruvate levels in cells following TSST-1 treatment. The results demonstrated a dose-dependent increase in the lactate-to-pyruvate ratio at both 24 and 48 hours with increasing TSST-1 concentrations, with the most significant increase observed at 1000 ng/mL, thus providing further evidence for TSST-1–induced metabolic reprogramming(Fig. 7 C). The elevated ratio reflects enhanced glycolytic flux under high-dose TSST-1 conditions. 4. Discussion TSST toxins have been previously reported to impair cell viability and metabolic processes, including glycolysis and ion transport [ 21 ]. Consistent with these findings, our data demonstrate that TSST-1 markedly decreases the viability of human vaginal epithelial cells in both dose- and time-dependent manners. In accordance with the critical roles of the G₁ phase in RNA synthesis and protein accumulation, as well as the S phase in DNA replication [ 22 ], treatment of HVEC with TSST at varying concentrations significantly induces apoptosis, reduces cell viability, and inhibits proliferation through cell cycle disruption. In our investigation, flow cytometric analysis further revealed rapid increases in apoptotic populations following TSST-1 exposure, along with G₁-phase accumulation and decreased S-phase fractions, suggesting a reduced proliferative capacity. These observations align with prior findings on TSST-mediated cytotoxicity [ 21 ], while expanding upon them by demonstrating a dose-graded pattern in vaginal epithelial cells, which has been inadequately addressed in previous research. RNA-seq revealed significant enrichment of multiple pathways associated with immunological and metabolic stress under high-dose conditions, including interferon signaling, glycolysis, ion transport, and structural remodeling The prominence of glycolysis and ion transport genes suggests that cells were metabolically adapting to inflammatory stress [ 23 , 24 ], likely engaging compensatory pathways to regulate intracellular pH amidst lactate accumulation. These pathways typically attenuate proliferative drive while reinforcing checkpoint inhibition; therefore, the observed G1-phase arrest in flow cytometry can be interpreted as a functional manifestation of energy and metabolic stress at the transcriptional level. Inflammatory stimulation can drive a metabolic shift from oxidative phosphorylation to glycolysis—a phenomenon known as the Warburg effect [ 25 ]. In alignment with this hypothesis, the observed increase in the lactate/pyruvate ratio in our work indicates that cells subjected to high TSST-1 undergo a Warburg-like shift, converting excess pyruvate to lactate rather than channeling it into mitochondrial oxidation. Meanwhile, mRNA expression of inflammatory cytokines, including IL-1β, IL-6, IL-8, MIP-α, TNF-α, and CXCL1 was markedly upregulated. For instance, IL-1β exposure has been shown to enhance glycolysis and increase lactate output in airway epithelial cells [ 26 ], while type I interferons can induce hexokinase 2 (HK2) and drive aerobic glycolysis in epithelial tissues. This demonstrates a distinct dose-dependent pattern: IL-1β and IL-6 peaked at moderate concentrations, whereas IL-8 and TNF-α continued to rise at higher doses. It is known that stimulation of genital tract epithelia induces the expression of antimicrobial peptides such as β-defensins and SLPI, which are regulated by pattern recognition receptors (PRRs) and hormonal signals, forming the epithelial effector arm [ 27 ]. The combined phenotype of increased apoptosis, cell-cycle arrest, and cytokine upregulation aligns with transcriptomic data showing significant enrichment of pro-apoptotic and stress-related pathways, indicating that inflammatory activation is mechanistically linked to early epithelial injury. At the transcriptomic level, RNA-seq findings further corroborated and expanded upon the aforementioned phenotypic changes. Compared to the control group, low-dose TSST-1 treatment led to the upregulation of 898 genes and downregulation of 581 genes, whereas high-dose treatment resulted in only 726 upregulated and 378 downregulated genes. This suggests that moderate stimulation induces broader and more functional transcriptional reprogramming, whereas extremely high doses drive cells into a stress-suppressed state [ 28 ]. GO/KEGG enrichment further demonstrated that low-dose responses were enriched in chemotaxis, antimicrobial defense, and receptor-ligand regulation, implying active immune recruitment, while high-dose treatment additionally triggered hypoxia-like metabolic stress and epithelial barrier remodeling. Although certain inflammatory cytokines were reduced in transcription within the cytokine–receptor interaction pathway, their receptor/ligand counterparts remained at elevated levels, suggesting negative feedback regulation rather than reduced functional output. These findings indicate that TSST-1 does not trigger a linear inflammatory response but exhibits threshold-dependent and pathway-specific regulatory characteristics, representing a mechanistic refinement compared to previous works. Previous studies have demonstrated that TSST exposure can alter gene expression and trigger cellular stress and apoptosis, specifically through NF-κB-mediated production of TNF-α, IL-6, and IL-8 [ 18 , 29 ]. Our KEGG data similarly revealed activation of inflammatory and apoptotic pathways, including cytokine–receptor interaction, chemokine signaling, TNF signaling, and stress-related networks such as HIF-1 (hypoxia) and glycolytic metabolism, supporting multiple converging axes of injury. HIF-1 enrichment implies reduced ATP production and suppression of the cell cycle [ 4 ]. IL-1β elevation further suggests IL-1 axis activation, consistent with reports showing IL-1β-mediated redistribution of ZO-1 and occludin, leading to epithelial barrier impairment [ 6 , 30 ]. Moreover, IL-1β can disrupt epithelial junctions via the NF-κB/MLCK axis, enhancing permeability [ 31 , 32 ]. Thus, our data together reinforce a mechanistic link between TSST-1, IL-1-driven inflammation, and barrier breakdown, which has not been systematically described previously in vaginal epithelial contexts. We also observed that the cellular effects of TSST-1 were not linearly cumulative across doses or timepoints but instead exhibited threshold and saturation behaviors, consistent with previous findings [ 33 ] [ 34 ]. IL-1β and IL-6 reached maximal induction at moderate doses, while IL-8 and TNF-α continued to increase, indicating separate activation thresholds and saturation dynamics. High toxin levels likely cause translation impairment, consistent with integrated stress response features in epithelial systems [ 35 – 37 ]. Reduced viability with preserved attachment during the first 24 h, followed by abrupt surges of apoptosis and detachment at 48–72 h, indicates the presence of a damage threshold–commitment point influenced by mitochondrial and checkpoint collapse [ 38 ]. Collectively, these results support a model in which TSST-1 not only activates inflammatory signaling but also simultaneously rewires cellular metabolism toward glycolysis to meet bioenergetic and biosynthetic demands—a Warburg-like reprogramming that may sustain inflammation but predispose cells to injury through lactate accumulation and metabolic acidosis [ 26 , 39 ]. Finally, our study delineated the early epithelial responses to TSST-1 from phenotype to transcriptome. However, the immortalized VK2/E6E7 line lacks the complete mucosal barrier and immune microenvironment. Previous reports indicate that VK2/E6E7 cells cultured at an air–liquid interface form multilayered, keratin-rich structures, with hormonal regulation significantly modulating barrier susceptibility [ 40 ]. Moreover, the observational time window of the present study was limited to 24–72 h. Future studies should employ primary epithelial cells or organoid models and incorporate pathway inhibition experiments targeting inflammation-related receptor signaling pathways and their roles in regulating glycolytic metabolism, in order to further validate the underlying molecular mechanisms and assess long-term metabolic consequences. 5. Conclusion This study characterized the early epithelial responses to TSST-1 at both cellular and molecular levels, uncovering a coordinated pattern of inflammatory amplification, cell-cycle arrest, apoptosis, and barrier perturbation. These findings enhance our understanding of the initial pathophysiological processes underlying TSS, particularly regarding the role of mucosal epithelia as the “frontline” linking local exposure to systemic inflammation. Our findings lay a theoretical foundation for evaluating the risk of TSS induced by vaginal TSST-1 exposure and offer practical directions for early intervention. Efforts may focus on developing molecular or antibody-based agents that directly neutralize TSST-1 and testing their protective efficacy in epithelial models; conversely, targeting the epithelial IL-1/NF-κB axes with pharmacologic or biologic modulators could mitigate toxin-induced cellular damage, offering a rational basis for early prevention and therapeutic strategies against TSS. Declarations Acknowledgements This work was partly funded by grants from the National Natural Science Fund of China (Grant No. 82150410452) and the Key Support Plan for Foreign Experts (Grant No. wgxz2022084L) to S.C.O. Author Contributions S.C.O. conceptualized the original research hypothesis, designed the project, and supervised this study. X.L. conducted the experiments, analyzed data, and drafted the manuscript. Y.Y. contributed to the methodology, data analysis, and project overview. C.K. provided assistance with resources, methods, and conceptual development. C.S. and H.Y. assisted with some methodology demonstration and provided analytical platforms. All authors reviewed the manuscript and approved the final version. Declaration of Competing Interest The authors declare no conflicts of interest. Data Availability The RNA sequencing data generated and analysed during the current study have been deposited in the NCBI BioProject database under accession number PRJNA1424937 . Other data supporting the findings of this study are included within the article and its supplementary materials. “Ethics, Consent to Participate, and Consent to Publish declarations: not applicable.” “Clinical trial number: not applicable.” References He Y et al. Transcriptome Analysis of Caco-2 Cells upon the Exposure of Mycotoxin Deoxynivalenol and Its Acetylated Derivatives. Toxins (Basel), 2021. 13(2). Shepherd FR, et al. The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12 – 4(+) memory T cells. Commun Biol. 2023;6(1):78. Wilkins AL, et al. 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RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. PLoS ONE. 2013;8(4):e61768. Liu T, et al. NF-kappaB signaling in inflammation. Signal Transduct Target Ther. 2017;2:17023. Kimura K, Teranishi S, Nishida T. Interleukin-1beta-induced disruption of barrier function in cultured human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2009;50(2):597–603. Kaminsky LW, Al-Sadi R, Ma TY. IL-1beta and the Intestinal Epithelial Tight Junction Barrier. Front Immunol. 2021;12:767456. Al-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight junction permeability. J Immunol. 2007;178(7):4641–9. Kimber I, et al. Toxic shock syndrome: characterization of human immune responses to TSST-1 and evidence for sensitivity thresholds. Toxicol Sci. 2013;134(1):49–63. Faulkner L, et al. The mechanism of superantigen-mediated toxic shock: not a simple Th1 cytokine storm. J Immunol. 2005;175(10):6870–7. Babon JJ, Varghese LN, Nicola NA. Inhibition of IL-6 family cytokines by SOCS3. Semin Immunol. 2014;26(1):13–9. Gao X, et al. Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells. BMC Cell Biol. 2007;8:17. Riesenberg BP, et al. Stress-Mediated Attenuation of Translation Undermines T-cell Activity in Cancer. Cancer Res. 2022;82(23):4386–99. Gupta S, et al. The mitochondrial death pathway: a promising therapeutic target in diseases. J Cell Mol Med. 2009;13(6):1004–33. Carreno-Florez GP, et al. Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection. PLoS Pathog. 2023;19(11):e1011719. Lee Y et al. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface. Viruses, 2016. 8(9). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8800664","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618710643,"identity":"62271562-6a05-4967-bf9b-911b9da36224","order_by":0,"name":"XUELING LUO","email":"","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"XUELING","middleName":"","lastName":"LUO","suffix":""},{"id":618710644,"identity":"adf6f8e2-fbc8-4433-b15b-72f87fc97ebc","order_by":1,"name":"yue yuan","email":"","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"yue","middleName":"","lastName":"yuan","suffix":""},{"id":618710645,"identity":"554fe19c-026d-42ba-bc9a-fdc1afe35026","order_by":2,"name":"ke chen","email":"","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"ke","middleName":"","lastName":"chen","suffix":""},{"id":618710646,"identity":"7adea4ac-bbc8-4b53-a202-40c51ba121e5","order_by":3,"name":"changfeng sun","email":"","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"changfeng","middleName":"","lastName":"sun","suffix":""},{"id":618710647,"identity":"dbd4cdfd-78b0-4118-8930-1e9ef6aec56b","order_by":4,"name":"hong yu","email":"","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"hong","middleName":"","lastName":"yu","suffix":""},{"id":618710648,"identity":"4669194f-52e3-4a16-90d2-7804b29e660c","order_by":5,"name":"Suvash Chandra Ojha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYLACxgYgnsHA+ADKNyBaCzNMKZFaGCQY2CSI0iI/I8fw488dDHLMs5ufVf5s25bYwN68TYKh5g5OLQY3coylec8wGDPOOWZ2m7ftdmIDz7EyCYZjz3BrkUhLkGZsY0hsnJFgdptxG1CLRI6ZBGPDYTwOS0v++ROsJf1b4U+QFvk3+LUw3Eg+JsEL1pJjxsALtoUHvxaDM4+PWQO1GDPOyCmW5v1327iNJ63YIuEYHoe1JzbfBDpMznBG+saPP87clu1nP7zxxocaPA6DgP8Mhg1QJhuISCCkAWwdMYpGwSgYBaNgZAIAvzZWonn8Ug8AAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital ofSouthwest Medical University","correspondingAuthor":true,"prefix":"","firstName":"Suvash","middleName":"Chandra","lastName":"Ojha","suffix":""}],"badges":[],"createdAt":"2026-02-05 20:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8800664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8800664/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106468157,"identity":"83f408a1-e953-47ea-971e-3e1740d7b703","added_by":"auto","created_at":"2026-04-09 00:29:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":556793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSDS-PAGE analysis of rTSST-1 expression, verification, and purification\u003c/strong\u003e\u003cbr\u003e\n (A) SDS-PAGE profile showing total protein before and after induction, and in crude lysates prior to purification. Lane M: molecular weight markers (kDa); Lane 1: total proteins from uninduced cells; Lane 2: total proteins from IPTG-induced cells; Lane 3: soluble fraction of lysed cells prior to chromatography. (B) SDS-PAGE analysis of Ni-NTA affinity-purified fractions shows a prominent band at the expected molecular weight, indicating effective enrichment of the His-fusion protein. (C) Western blot analysis using an anti-His antibody specifically detected a single immunoreactive band at the expected molecular weight in the induced (Lane 2) and Ni-NTA–purified sample (Lane 3), whereas no signal was observed in the uninduced control (Lane 1). Lane M represents molecular weight markers in kDa. (D) SDS-PAGE after removal of the fusion/His tag exhibited a sharp single band with minimal background (Lane 1), thereby confirming successful cleavage.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/ca287421c8cb76d515e1c716.png"},{"id":107480189,"identity":"849f4e39-a21b-4efa-8c22-b42cb0e5f099","added_by":"auto","created_at":"2026-04-22 02:06:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1169328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of TSST-1 on the viability and proliferation of HVEC\u003c/strong\u003e\u003cbr\u003e\n (A) CCK-8 time-course curves of cell viability (0–96 h) after treatment with rTSST-1 (0–1000 ng/mL). (B) Bar charts show cell viability at 24, 48, 72, and 96 h, expressed as a percentage of the untreated control (mean ± SD, n=3). (C) EdU staining images show proliferating nuclei (EdU⁺, red) and total nuclei (Hoechst, blue). (D) Quantification of EdU-positive areas at different concentrations. (E) Phase-contrast micrographs show cell morphological changes in response to increasing TSST-1 concentrations and prolonged exposure.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/e8449d0b771af0d220c932e3.png"},{"id":106725048,"identity":"c82827ba-1049-41b4-8187-d2b96c3ab5a1","added_by":"auto","created_at":"2026-04-12 18:31:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":693680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSST-1 induces apoptosis and G₁-phase arrest in HVEC\u003c/strong\u003e\u003cbr\u003e\n (A) Flow cytometric analysis using Annexin V-FITC/PI double staining, showing changes in early and late apoptotic cell populations following treatment with increasing concentrations of TSST-1. The right panel presents quantitative apoptosis rates (mean ± SD, n=3). (B) Calcein-AM/PI live–dead cell staining images, where green fluorescence indicates live cells and red fluorescence indicates dead cells, with corresponding quantitative analysis on the right. (C) Cell-cycle distribution histograms after exposure to different TSST-1 concentrations for 24, 48, and 72 h. With increasing toxin concentrations, the proportion of cells in the G₁ phase increased, while the S-phase fraction decreased, suggesting cell-cycle arrest. Collectively, these results demonstrate that TSST-1 markedly promotes apoptosis and induces G₁-phase blockade, thereby suppressing epithelial cell proliferation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/4120484cceb03c09ba5c7c19.png"},{"id":106725836,"identity":"e0f936a8-5c20-4140-b606-63b49aff4263","added_by":"auto","created_at":"2026-04-12 18:34:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSST-1 induces upregulation of proinflammatory cytokine expression in HVEC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative RT-qPCR measured the mRNA expression levels of inflammation-related genes: (A) IL-1β, (B) IL-6, (C) IL-8, (D) MIP-α, (E) TNF-α, and (F) CXCL1 following treatment with TSST-1 at concentrations of 0, 500, and 1000 ng/mL, with the LPS group serving as a positive control. Results are expressed as fold change relative to the control group (mean ± SD, n = 3). TSST-1 significantly upregulated the transcription levels of multiple proinflammatory genes, indicating its ability to activate the inflammatory response pathways in vaginal epithelial cells.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/6e1d066ae1fe952b7638f605.png"},{"id":106468160,"identity":"55aaa64d-620a-41bc-a191-cde6ee06d9ef","added_by":"auto","created_at":"2026-04-09 00:29:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":644258,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic heterogeneity and functional responses triggered by TSST-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Volcano plots showing differentially expressed genes (DEGs) in low-dose (125 ng/mL) and high-dose (1000 ng/mL) TSST-1 treatments relative to control (NC); red indicates upregulation, while blue denotes downregulation. (B) A Venn diagram and bar chart displaying the overlap and unique DEGs among the two treatment groups and the control group. (C) Principal component analysis (PCA) showing obvious distinction among the three groups, indicating pronounced transcriptional divergence following TSST-1 treatment. (D) GO enrichment bubble plot illustrating significantly altered biological processes, including chemotaxis, antimicrobial defense, and receptor–ligand modulation.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/827f3f07aa45b17c537e656b.png"},{"id":106725892,"identity":"03c721c8-50e7-4f3f-937f-e7c70471c681","added_by":"auto","created_at":"2026-04-12 18:34:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":589455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway enrichment, key gene expression, and PPI network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) KEGG enrichment bubble plot: the low-dose group was mainly enriched in “cytokine–cytokine receptor interaction,” “chemokine signaling,” and classical immune pathways, including IL-17, TLR, and NOD-like receptor signaling; the high-dose group exhibited additional involvement in PI3K-Akt, VEGF, HIF-1, glycolysis/gluconeogenesis, arginine–proline metabolism, and PPAR pathways. (B) Bar charts illustrating the quantitative expression (log₁₀(FPKM+1)) of genes in the cytokine–cytokine receptor interaction pathway, alongside corresponding qPCR results wherever applicable. A majority of chemokine and interleukin genes were downregulated in the treated groups, while a few receptors or ligands showed slight upregulation. (C) Protein–protein interaction (PPI) network illustrating key regulatory nodes and modular rearrangements under low- and high-dose conditions, highlighting a dose-dependent network reorganization centered on the cytokine–chemokine axis.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/9dc844cc446ff23aa8e6fcde.png"},{"id":106724483,"identity":"f9143a1f-d9d0-4970-96ec-dbd0d342a0e7","added_by":"auto","created_at":"2026-04-12 18:28:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":572448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSST-1 induces dose-dependent glycolytic reprogramming in HVEC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in the TSST-high and TSST-low groups. High-dose TSST-1 treatment was enriched in pathways related to interferon signaling, GPCR signaling, and potassium (K⁺) channel activity, while low-dose treatment primarily enriched pathways related to ion transport and neuronal processes. (B) Heatmaps of representative DEGs. The TSST-high group showed marked upregulation of IFIT1/2/3, OASL, HK2, CA9, and KRT16, whereas the TSST-low group specifically upregulated CXCL3, PPP1R14A, NECTIN4, TXNIP, and G0S2. (C) The lactate/pyruvate ratio in culture supernatants elevated with increasing TSST-1 concentration at both 24 h and 48 h, exhibiting a notable peak at 1000 ng/mL.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/f5f7254ceee81fe89c6e5271.png"},{"id":107482274,"identity":"11f2de19-d78b-4d1f-b666-a47aad4babeb","added_by":"auto","created_at":"2026-04-22 02:22:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4316092,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/4b72d4ec-da9f-467f-8255-058086495b68.pdf"},{"id":106468156,"identity":"ba02d0a3-5633-40ed-bc36-c89a2713db05","added_by":"auto","created_at":"2026-04-09 00:29:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":763536,"visible":true,"origin":"","legend":"","description":"","filename":"supplymentfilenew.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8800664/v1/02ab97f357ffe1e0fcbb32cd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptomic Profiling of TSST-1–Induced Cytotoxicity and Inflammatory Responses in Human Vaginal Epithelial Cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eToxic shock syndrome (TSS) is a life-threatening systemic disorder triggered by superantigens (SAgs) secreted by certain \u003cem\u003eStaphylococcus aureus\u003c/em\u003e or \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinically, TSS typically begins with transient, nonspecific prodromal symptoms and can rapidly progress to hypotension, shock, and multiorgan failure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among staphylococcal superantigens, toxic shock syndrome toxin-1 (TSST-1) is the prototypical virulence factor: it binds major histocompatibility complex class II (MHC II) molecules on antigen-presenting cells and crosslinks them with the Vβ domain of T-cell receptors, bypassing conventional antigen processing and driving massive, non-specific T-cell activation with excessive release of TNF-α, IL-1, IL-2, IFN-γ, and other mediators, culminating in a cytokine storm [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Epidemiological observations in the 1980s, when TSST-1\u0026ndash;associated menstrual TSS rose sharply in parallel with high-absorbency tampon use, further pointed to the female reproductive tract as a critical portal of exposure; risk was associated with prolonged single-tampon wear, overnight use, and suboptimal insertion practices [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this context, the vaginal epithelium is not only a physical barrier but also an immunologically active interface that produces antimicrobial peptides, transports IgA, secretes chemokines, and engages in hormone-dependent (e.g., estrogen-regulated) crosstalk with local and systemic immunity[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClinically, current management strategies focus on rapid control of infection and containment of systemic consequences, including empirical broad-spectrum antibiotics, source control via imaging-guided intervention or surgical debridement when needed, and prompt removal of foreign materials such as tampons, nasal packing, or wound dressings [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Combination regimens that pair β-lactams with clindamycin are commonly used, as clindamycin suppresses bacterial protein synthesis and reduces superantigen production [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, no approved therapy directly neutralizes TSST-1, and existing approaches primarily target bacterial load or systemic inflammation rather than the initial mucosal events. This gap underscores the need to refocus attention on the vaginal mucosa as a frontline site where TSST-1 is sensed, where barrier integrity may be compromised, and where early inflammatory programming could shape downstream systemic responses. As part of this frontline, tight junction proteins such as occludin, claudin-1, and ZO-1 are essential for barrier maintenance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while pattern recognition receptors (PRRs) enable epithelial cells to detect microbial and toxin-associated signals and rapidly secrete TNF-α, IL-1β, IL-6, chemokines, and antimicrobial peptides to recruit neutrophils and restrict bacterial dissemination [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite extensive work on TSST-1\u0026ndash;induced systemic immune dysregulation and cytokine storm, direct evidence delineating its early impact on human vaginal epithelial cells (HVEC) remains limited. Prior \u003cem\u003ein vitro\u003c/em\u003e studies have often focused on single endpoints such as isolated cytokine readouts or barrier-related markers without integrating cytotoxicity, proliferative capacity, cell-cycle dynamics, apoptosis, and inflammatory signaling into a unified framework [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To address this knowledge gap, we employed the HVEC line VK2/E6E7 and exposed it to graded concentrations of recombinant TSST-1 under time-controlled conditions, with the aim of answering three interrelated questions: (i) whether TSST-1 induces measurable cytotoxicity in a dose- and time-dependent manner; (ii) whether the balance between proliferation and programmed cell death shifts, reflected by changes in apoptosis rates and cell-cycle distribution; and (iii) whether epithelial inflammatory programs are activated at both the transcriptional and protein levels.\u003c/p\u003e \u003cp\u003eHere, we integrated metabolic and growth assays to evaluate cell viability, employed flow cytometry to analyze apoptosis and cell-cycle status, and quantified representative cytokines at both mRNA and secretory levels, while correlating these phenotypic data with RNA-seq and pathway enrichment analyses to map TSST-1\u0026ndash;responsive signaling networks. By linking epithelial-level cytotoxicity, proliferative restraint, and apoptosis with transcriptional remodeling of barrier- and immune-related pathways, this study provides a mucosal perspective that helps connect local epithelial perturbations to the broader systemic dysregulation observed in TSS, thereby offering a basis for earlier detection and targeted intervention strategies. Simultaneously, emerging evidence indicates that anti-virulence compounds targeting the \u003cem\u003eS. aureus\u003c/em\u003e SaeRS two-component system can suppress TSST-1 production without exerting strong bactericidal pressure, thereby supporting the feasibility of interventions aimed at modulating toxin regulation rather than solely relying on conventional antibiotics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], reinforcing the relevance of dissecting TSST-1\u0026rsquo;s direct effects on vaginal epithelial cells.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Construction of His-tagged Recombinant Plasmid\u003c/h2\u003e \u003cp\u003eThe TSST-1 gene sequence was retrieved from NCBI with the GenBank accession number J02615. The synthetic TSST-1 gene sequence, featuring a deleted stop codon and incorporating BamH1 and XhoI restriction sites at the 5' and 3' ends, respectively, was introduced into the pET28a(+) expression vector (SciProtech, Beijing) as a fusion with a His-tag (H\u003csub\u003e6\u003c/sub\u003e) and SUMO (small ubiquitin-like modifier). The sequence of the recombinant plasmid (TSST-H6-SUMO-pET28a (+)) was validated using restriction digestion and DNA sequencing to ensure its integrity and the absence of mutations, and it was subsequently transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e NiCo21 (DE3) for expression and purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Protein Expression and Verification\u003c/h2\u003e \u003cp\u003eA single colony of \u003cem\u003eE. coli\u003c/em\u003e NiCo21 (DE3) harboring the \u003cem\u003etsst\u003c/em\u003e expression plasmid was inoculated into 10 mL of LB medium and cultured overnight at 37\u0026deg;C with shaking (220 rpm). The culture was diluted 1:100 into 500 mL of fresh LB medium and grown to mid-log phase (OD₆₀₀ ~0.6). Protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) and incubated for 20 h at 37\u0026deg;C. Cells were harvested by centrifugation (9000 rpm, 4\u0026deg;C, 30 min), resuspended in RIPA buffer (Beyotime, P0013B), and lysed on ice for 30 min with gentle intermittent vortexing. Following clarification of the lysate by centrifugation (12 000 rpm, 4\u0026deg;C, 15 min), the resultant supernatants, representing the soluble protein fractions, were analyzed by 12% SDS-PAGE alongside uninduced controls to confirm expression and solubility.\u003c/p\u003e \u003cp\u003eAdditionally, the identity and tag status of the resulting protein were subsequently verified by Western blotting. Briefly, samples resolved by SDS\u0026ndash;PAGE were electrotransferred onto a nitrocellulose membrane, blocked with a protein-free rapid blocking solution (PS108P, EpiZyme) at room temperature, and incubated with a mouse anti-His tag monoclonal antibody (AE003, Abclonal), followed by a goat anti-mouse IgG secondary antibody (Servicebio). Immunoreactive bands were detected using an enhanced chemiluminescence (ECL) detection system (Omni-ECL\u0026trade;, EpiZyme) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Protein Purification\u003c/h2\u003e \u003cp\u003eFiltered supernatants were loaded onto an affinity-based Ni\u0026sup2;⁺-NTA gravity column (5-mL HisTrap\u0026trade; HP) pre-equilibrated with Buffer A (20 mM phosphate buffer, pH 7.4, 0.5 M NaCl, 0.05% Tween-20, 20 mM imidazole). The column was washed with 5\u0026ndash;10 column volumes (CV) of Buffer A before being eluted stepwise with 50, 100, 250, and 500 mM imidazole (each 5 CV) from Buffer B (500 mM imidazole). Protein fractions were screened by 12% SDS-PAGE. The 50 mM eluate was treated with ubiquitin-like-specific protease (ULP1, ~\u0026thinsp;10 U per 50 \u0026micro;g protein) at 30\u0026deg;C for 2 h to remove the SUMO tag, then reloaded onto the Ni\u0026sup2;⁺-NTA column to collect flow-through fractions. The final fractions were pooled for anion-exchange (Q) chromatography following sequential 20 mM and 500 mM imidazole wash/elution cycles. Except for protease digestion at 30\u0026deg;C, all steps were performed at 4\u0026deg;C to prevent degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 His-tag Removal and Post-Cleavage Purification\u003c/h2\u003e \u003cp\u003ePurified His-tagged rTSST-1 was transferred into cleavage buffer and digested with site-specific protease at the engineered linker to remove the N-terminal His tag. Aliquots were periodically collected, mixed with 2\u0026times; SDS loading buffer to terminate the reaction, and analyzed by reducing 12% SDS-PAGE. After complete digestion, the sample was buffer-exchanged into phosphate buffer (pH 7.4) containing 20 mM imidazole and 0.5 M NaCl, and was subsequently reloaded into a Ni\u0026sup2;⁺-NTA column. The untagged rTSST-1 was collected in the flow-through and low-imidazole wash fractions, while residual His-tagged and any uncleaved proteins were retained on the matrix. All chromatographic steps were conducted at 4\u0026deg;C, and fractions containing the target proteins were verified by SDS-PAGE, yielding homogenous tag-free rTSST-1 for subsequent assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Culture\u003c/h2\u003e \u003cp\u003eThe HVEC (VK2/E6E7) was purchased from iCell. Cells were maintained in serum-free VK2/E6E7 medium (iCell-h551-001b, 500 mL) supplemented with the manufacturer\u0026rsquo;s recommended growth factors, 1% FBS (SPERIKON SP010010500) and 1% penicillin/streptomycin. This medium formulation was used for both routine culture and subsequent toxin exposure experiments. Cultures were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂. Upon reaching 80% confluence, cells were passaged using 0.25% trypsin-EDTA (HyClone SH30042.01) for approximately 4 min at 37\u0026deg;C. Trypsinization was halted by adding medium containing 5% FBS or through centrifugation and resuspension in fresh medium. Cells from early passages (P5\u0026ndash;P15) were employed for all experiments. The culture medium was replaced 2 to 3 times per week, and cells were passaged at ratios of 1:2 to 1:5 every 4 to 6 days [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell Viability Assay\u003c/h2\u003e \u003cp\u003eVK2/E6E7 cells were seeded at 3000 cells/well in 96-well plates and pre-cultured for 24 h. Cells were then treated with rTSST-1 at final concentrations of 0, 60, 125, 250, 500, and 1000 ng/mL for 24, 48, 72, and 96 h. Each condition was performed in five technical replicates across three distinct biological experiments, with wells randomized across plates. Following treatment, 10 \u0026micro;L of CCK-8 reagent (GLPBIO GK10001-30) was added to each well, plates were incubated for 2 h at 37\u0026deg;C, and absorbance was measured at 450 nm. The viability of each treatment group was standardized to the untreated control (0 ng/mL) and is expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Cell Proliferation Assay\u003c/h2\u003e \u003cp\u003eCell proliferation was assessed using EdU labeling. VK2/E6E7 cells were seeded in 24-well plates and treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 48 h. Pre-warmed 2\u0026times; EdU working solution was added to a final concentration of 10 \u0026micro;M and incubated for 4 h. Subsequently, cells were fixed with 4% paraformaldehyde (15 min), permeabilized with 0.3% Triton X-100/PBS (15 min), and stained using Azide-594 click reaction (30 min) in the dark. Nuclei were counterstained with Hoechst 33342 for 10 min. Fluorescence images were captured (Ex/Em 590/615 nm) and the proliferation rate (EdU⁺/Hoechst⁺ ratio) was assessed from a minimum of five fields per well across two independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Apoptosis Detection Assay\u003c/h2\u003e \u003cp\u003eApoptosis was quantified using the BD Pharmingen\u0026trade; FITC Annexin V Apoptosis Detection Kit (556547). VK2/E6E7 cells in 6-well plates were treated with 0, 250, 500, or 1000 ng/mL TSST-1 for 48 h. Following two washes with cold PBS, 1\u0026times;10⁵ cells were resuspended in 100 \u0026micro;L of 1\u0026times; binding buffer, stained with 5 \u0026micro;L of FITC Annexin V and 5 \u0026micro;L of PI, and incubated for 15 min at room temperature in the dark. After adding 400 \u0026micro;L of binding buffer, samples were analyzed within 1 h on a BD FACSAria cytometer. Triplicate biological replicates were processed, and populations (live, early apoptotic, and late apoptotic/necrotic) were quantified using FlowJo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Live/dead Cell Staining\u003c/h2\u003e \u003cp\u003eLive/dead ratios were visualized by Calcein AM/PI staining (Beyotime, C2015M). VK2/E6E7 cells in 24-well plates were treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 48 h, washed once with PBS, and incubated with 250 \u0026micro;L of Calcein AM/PI working solution at 37\u0026deg;C for 30 min in the dark. Images were captured on an Olympus IX73 microscope using green (Calcein AM, Ex/Em 494/517 nm) and red (PI, Ex/Em 535/617 nm) channels. Two biological repeats were performed, analyzing\u0026thinsp;\u0026ge;\u0026thinsp;5 random fields per well for Calcein⁺ and PI⁺ cell percentages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cell Cycle Analysis\u003c/h2\u003e \u003cp\u003eVK2/E6E7 cells were treated with TSST-1 (0-1000 ng/mL) for 72 h, subsequently harvested, and fixed overnight in 75% ethanol at \u0026minus;\u0026thinsp;20\u0026deg;C. Following washing, cells were stained with 0.5 mL of PI/RNase solution (BD Pharmingen) for 15 min at room temperature in the dark, then stored at 4\u0026deg;C and analyzed within 1 h using a BD FACSAria cytometer (BD Biosciences, instrument ID TY2013001909). Modfit was used to compute cell-cycle distributions (G₀/G₁, S, G₂/M) across three independent biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Quantitative Real-Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells following a 24 h treatment with TSST-1 (0, 500, 1000 ng/mL) or 1000 ng/mL LPS (positive control) using a commercial kit (TIANGEN DP419). The purity and integrity of RNA were assessed using NanoDrop and agarose gel electrophoresis, respectively. One microgram of RNA was reverse-transcribed using SweScript All-in-One RT SuperMix (Servicebio G3337)- Quantitative PCR (qPCR) was performed in triplicate technical wells on a QuantStudio 3 system employing 2\u0026times; SYBR Green Master Mix (High ROX, Servicebio G3322-05) under the following cycling program: 95\u0026deg;C for 30 s; 40 cycles of 95\u0026deg;C for 15 s, 60\u0026deg;C for 10 s, 72\u0026deg;C for 30 s; followed by melting curve analysis. The targets included IL-1β, IL-6, IL-8, CCL3, TNF, and CXCL1, with β-actin serving as an internal control. Relative expression was determined using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt technique.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 RNA-seq\u003c/h2\u003e \u003cp\u003eVK2/E6E7 cells were treated with 125 ng/mL (low) or 1000 ng/mL (high) of rTSST-1 for 48 h, while untreated cells served as negative controls (NC), and were processed in triplicate. Total RNA was extracted using TRIzol and subsequently submitted to Novogene for library construction and sequencing. Raw FASTQ reads were cleaned with SeqPrep and Sickle, then aligned to the reference genome using Tophat2. Gene/transcript expression was quantified with HTSeq, and differential expression was analyzed by DESeq with a threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify pathways modulated by TSST-1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Lactate/Pyruvate Ratio Assay\u003c/h2\u003e \u003cp\u003eThe lactate-to-pyruvate (L/P) ratio was quantified in triplicate using a commercial enzymatic assay kit (Servicebio), following the manufacturer's protocols. Briefly, supernatants from VK2/E6E7 cells treated with TSST-1 (0, 250, 500, 1000 ng/mL) for 24 or 48 h were collected. To quantify lactate, 50 \u0026micro;L of each sample was combined with 44 \u0026micro;L of lactate assay buffer, 2 \u0026micro;L of Amplex Red, 2 \u0026micro;L of Enzyme Solution A, and 2 \u0026micro;L of Enzyme Solution B to prepare a 100 \u0026micro;L reaction mixture. To test pyruvate, 72 \u0026micro;L of pyruvate assay buffer, 2 \u0026micro;L of Amplex Red, 2 \u0026micro;L of Enzyme Solution A, 2 \u0026micro;L of Enzyme Solution B, and 2 \u0026micro;L of cofactor were combined to make an 80 \u0026micro;L working solution. All reactions were incubated in the dark for 30 minutes at room temperature, and absorbance was measured at 560 nm. The final L/P ratios were derived using standard curves and normalized to the control group (0 ng/mL), with data expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were independently performed at least three times, and the data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The statistical significance between two groups was evaluated using a two-tailed Student\u0026rsquo;s t-test, whereas comparisons across multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s or Dunnett\u0026rsquo;s post hoc test, as applicable. For data that did not satisfy the assumptions of normality or homogeneity of variance, corresponding nonparametric tests were applied. Statistical analyses were performed using GraphPad Prism 9.0 software, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Differentially expressed genes (DEGs) in transcriptomic data were identified using the DESeq2 package with thresholds of |log₂FC| \u0026ge; 1 and adjusted q\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Benjamini\u0026ndash;Hochberg correction).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Soluble Expression, Verification and Purification of rTSST-1\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) SDS-PAGE profile showing total protein before and after induction, and in crude lysates prior to purification. Lane M: molecular weight markers (kDa); Lane 1: total proteins from uninduced cells; Lane 2: total proteins from IPTG-induced cells; Lane 3: soluble fraction of lysed cells prior to chromatography. (B) SDS-PAGE analysis of Ni-NTA affinity-purified fractions shows a prominent band at the expected molecular weight, indicating effective enrichment of the His-fusion protein. (C) Western blot analysis using an anti-His antibody specifically detected a single immunoreactive band at the expected molecular weight in the induced (Lane 2) and Ni-NTA\u0026ndash;purified sample (Lane 3), whereas no signal was observed in the uninduced control (Lane 1). Lane M represents molecular weight markers in kDa. (D) SDS-PAGE after removal of the fusion/His tag exhibited a sharp single band with minimal background (Lane 1), thereby confirming successful cleavage.\u003c/p\u003e \u003cp\u003eFor successful application of TSST-1 protein in cellular assays, the r\u003cem\u003eTSST-1\u003c/em\u003e gene was cloned into the pET-28a(+) vector to generate an N-terminal His-tagged construct. This construct was expressed in \u003cem\u003eE. coli\u003c/em\u003e under IPTG induction and purified using Ni-NTA affinity chromatography, followed by site-specific protease cleavage and re-chromatography to remove the His tag. The expression, solubility, purification efficiency, and tag removal of rTSST-1 were then verified, and each stage was examined using SDS-PAGE. No distinct band was observed at the target molecular weight in uninduced samples, whereas a prominent band appeared at approximately 39 kDa after IPTG induction and remained detectable in the soluble fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating predominantly soluble expression. Ni-NTA affinity purification yielded a single\u0026thinsp;~\u0026thinsp;39 kDa band with minimal impurities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For further validation, purified rTSST-1 was analyzed by Western blotting using an anti-His antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). A clear immunoreactive band was detected at approximately 39 kDa in lanes 2 and 3, whereas no specific signal was observed in lane 1, confirming successful expression and purification of His-tagged rTSST-1 with high specificity. Following tag removal, the major band shifted to ~\u0026thinsp;26 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), which corresponds to the expected theoretical molecular weight of tag-free TSST-1, confirming complete cleavage and intact protein integrity. These results demonstrate that recombinant TSST-1 was successfully expressed in soluble form, purified to high homogeneity, and effectively de-tagged, meeting the requirements for subsequent cellular experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 TSST-1 Inhibits the Viability and Proliferation of Vaginal Epithelial Cells in a Time- and Dose-Dependent Manner\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) CCK-8 time-course curves of cell viability (0\u0026ndash;96 h) after treatment with rTSST-1 (0\u0026ndash;1000 ng/mL). (B) Bar charts show cell viability at 24, 48, 72, and 96 h, expressed as a percentage of the untreated control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3). (C) EdU staining images show proliferating nuclei (EdU⁺, red) and total nuclei (Hoechst, blue). (D) Quantification of EdU-positive areas at different concentrations. (E) Phase-contrast micrographs show cell morphological changes in response to increasing TSST-1 concentrations and prolonged exposure.\u003c/p\u003e \u003cp\u003eTo further assess the effects of TSST-1 on VK2/E6E7 cells, viability (CCK-8), proliferation (EdU), and morphological alterations were analyzed over a period of 96 h. Compared to the control, the CCK-8 curves revealed a gradual decline in viability with increasing toxin concentration and prolonged exposure: with the exception of the highest dose (1000 ng/mL), all treatment groups showed diminished increases over time, remaining consistently below control levels, with widening disparities observed between 24 and 96 h. The 1000 ng/mL curve consistently exhibited the lowest and was virtually flat throughout (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Bar-plot analysis showed that at 24 h, only the high-dose group exhibited a noticeable drop; at 48 h, viability suppression became significant from mid to high doses, and by 72\u0026ndash;96 h, all doses displayed distinct and graded declines (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). EdU staining confirmed this trend: red EdU⁺ nuclei decreased progressively with higher toxin levels, and merged images revealed a marked reduction of the red\u0026ndash;blue overlap region. The quantitative analysis of EdU⁺ areas showed consistent, statistically significant dose-dependent reductions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eUnder phase-contrast microscopy, cells transitioned from densely adherent and dispersed morphologies to rounder, shrunken forms with widened intercellular spaces and partial detachment. These morphological changes intensified from 24 h to 96 h, most prominently in the high-dose group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these results demonstrate that TSST-1 exerts both time- and concentration-dependent inhibitory effects on epithelial cell viability and proliferation, along with progressive morphological deterioration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 TSST-1 Induces Enhanced Apoptosis and Disrupts the Cell Cycle\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Flow cytometric analysis using Annexin V-FITC/PI double staining, showing changes in early and late apoptotic cell populations following treatment with increasing concentrations of TSST-1. The right panel presents quantitative apoptosis rates (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3). (B) Calcein-AM/PI live\u0026ndash;dead cell staining images, where green fluorescence indicates live cells and red fluorescence indicates dead cells, with corresponding quantitative analysis on the right. (C) Cell-cycle distribution histograms after exposure to different TSST-1 concentrations for 24, 48, and 72 h. With increasing toxin concentrations, the proportion of cells in the G₁ phase increased, while the S-phase fraction decreased, suggesting cell-cycle arrest. Collectively, these results demonstrate that TSST-1 markedly promotes apoptosis and induces G₁-phase blockade, thereby suppressing epithelial cell proliferation.\u003c/p\u003e \u003cp\u003eTo further substantiate the cellular effects of TSST-1, apoptosis (Annexin V/PI), live/dead cell ratios (Calcein-AM/PI), and cell-cycle distribution were analyzed after 24 h of treatment. In comparison to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), early and late apoptotic populations increased notably at 250, 500, and 1000 ng/mL, with apoptosis rates rising dose-dependently and achieving statistical significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Calcein-AM/PI staining revealed a gradual reduction of viable (green) cells and a corresponding rise in dead (red, PI-positive) cells with increasing toxin concentration, consistent with quantitative analysis showing a significant dose-dependent decline in viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, cell-cycle analysis indicated that increased TSST-1 exposure boosted the proportion of cells in the G\u003csub\u003e1\u003c/sub\u003e phase while reducing those in the S phase, with both changes strongly correlated with concentration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), implying cell-cycle arrest. These results collectively demonstrate that TSST-1 markedly promotes apoptosis and induces G₁-phase arrest, thereby suppressing epithelial cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Dose-dependent Activation of Epithelial Proinflammatory Responses by TSST-1\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantitative RT-qPCR measured the mRNA expression levels of inflammation-related genes: (A) IL-1β, (B) IL-6, (C) IL-8, (D) MIP-α, (E) TNF-α, and (F) CXCL1 following treatment with TSST-1 at concentrations of 0, 500, and 1000 ng/mL, with the LPS group serving as a positive control. Results are expressed as fold change relative to the control group (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3). TSST-1 significantly upregulated the transcription levels of multiple proinflammatory genes, indicating its ability to activate the inflammatory response pathways in vaginal epithelial cells.\u003c/p\u003e \u003cp\u003eTo evaluate the proinflammatory response induced by TSST-1, we performed qPCR validation and quantitative analysis of key cytokine and chemokine genes involved in inflammatory signalling pathways, including IL-1β, IL-6, IL-8, TNF, CXCL1, and CCL3 (MIP-α). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the y-axis denoting fold change, with groups consisting of 0, 500, and 1000 ng/mL TSST-1, as well as LPS. Compared to the untreated control, IL-1β and IL-6 increased significantly at 500 ng/mL and remained elevated at 1000 ng/mL. However, there was a \u0026ldquo;low-dose higher than high-dose\u0026rdquo; trend, demonstrating a non-linear pattern. In contrast, IL-8, MIP-α, TNF-α, and CXCL1 showed little or no change at 500 ng/mL but were significantly upregulated at 1000 ng/mL, with overall expression levels comparable to or exceeding those of the LPS positive control. These results indicate that TSST-1 exhibits a graded response among different inflammatory factors: IL-1β and IL-6 reach their maximum at lower doses, whereas IL-8, MIP-α, TNF-α, and CXCL1 increase with higher concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Transcriptomic Profiling Reveals Core Response Pathways and Dose-dependent Regulation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) Volcano plots showing differentially expressed genes (DEGs) in low-dose (125 ng/mL) and high-dose (1000 ng/mL) TSST-1 treatments relative to control (NC); red indicates upregulation, while blue denotes downregulation. (B) A Venn diagram and bar chart displaying the overlap and unique DEGs among the two treatment groups and the control group. (C) Principal component analysis (PCA) showing obvious distinction among the three groups, indicating pronounced transcriptional divergence following TSST-1 treatment. (D) GO enrichment bubble plot illustrating significantly altered biological processes, including chemotaxis, antimicrobial defense, and receptor\u0026ndash;ligand modulation.\u003c/p\u003e \u003cp\u003eThe transcriptomic effects of TSST-1 were meticulously investigated by comparing RNA-seq profiles of the control (NC), low-dose (TSST low), and high-dose (TSST high) groups. Relative to the control, the TSST-low group exhibited 898 upregulated and 581 downregulated genes, whereas the TSST-high group showed 726 upregulated and 378 downregulated genes, indicating that TSST-1 elicited a broader transcriptional response at a lower toxin concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The Venn diagram analysis demonstrated a substantial overlap of differentially expressed genes between the two treatment groups, while each condition retained distinct DEG subsets, indicating the coexistence of conserved core transcriptional responses alongside dose-dependent variations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Principal component analysis revealed distinct spatial separation among NC, TSST-low, and TSST-high samples, with tight clustering of biological replicates within each group, indicating pronounced transcriptional divergence following TSST-1 stimulation and supporting the reproducibility and robustness of the sequencing data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Functional enrichment analysis employing Gene Ontology showed that both TSST-low and TSST-high groups were predominantly enriched in immune-related biological processes, including receptor\u0026ndash;ligand regulation, antiviral defense, and chemotactic signaling. In contrast, the high-dose condition further enriched stress-associated terms such as hypoxia response, cytoskeletal organization, and ion channel\u0026ndash;related activities, suggesting a shift from immune activation to cellular stress and functional remodeling at elevated TSST-1 concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) KEGG enrichment bubble plot: the low-dose group was mainly enriched in \u0026ldquo;cytokine\u0026ndash;cytokine receptor interaction,\u0026rdquo; \u0026ldquo;chemokine signaling,\u0026rdquo; and classical immune pathways, including IL-17, TLR, and NOD-like receptor signaling; the high-dose group exhibited additional involvement in PI3K-Akt, VEGF, HIF-1, glycolysis/gluconeogenesis, arginine\u0026ndash;proline metabolism, and PPAR pathways. (B) Bar charts illustrating the quantitative expression (log₁₀(FPKM\u0026thinsp;+\u0026thinsp;1)) of genes in the cytokine\u0026ndash;cytokine receptor interaction pathway, alongside corresponding qPCR results wherever applicable. A majority of chemokine and interleukin genes were downregulated in the treated groups, while a few receptors or ligands showed slight upregulation. (C) Protein\u0026ndash;protein interaction (PPI) network illustrating key regulatory nodes and modular rearrangements under low- and high-dose conditions, highlighting a dose-dependent network reorganization centered on the cytokine\u0026ndash;chemokine axis.\u003c/p\u003e \u003cp\u003eKEGG analysis was consistent with these observations. The low-dose group was enriched in \u0026ldquo;cytokine\u0026ndash;cytokine receptor interaction,\u0026rdquo; \u0026ldquo;chemokine signaling,\u0026rdquo; and classical immune pathways, including IL-17, TLR, and NOD-like receptor signaling, whereas the high-dose group extended to PI3K-Akt, VEGF, HIF-1, glycolysis/gluconeogenesis, arginine\u0026ndash;proline metabolism, and PPAR pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To visualize changes in immune- and chemokine-related genes more intuitively, a bar chart of log\u003csub\u003e10\u003c/sub\u003e(FPKM\u0026thinsp;+\u0026thinsp;1) values was generated. Most chemokine and interleukin genes (e.g., \u003cem\u003eCXCL2\u003c/em\u003e, \u003cem\u003eCXCL3\u003c/em\u003e, \u003cem\u003eCXCL10\u003c/em\u003e, \u003cem\u003eIL24\u003c/em\u003e, \u003cem\u003eIL1RN\u003c/em\u003e) showed reduced expression compared to the control, while a few receptors or ligands were slightly upregulated, suggesting potential feedback regulation within the overall inflammatory activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Simultaneously, the corresponding PPI network showed that key nodes clustered around the cytokine\u0026ndash;chemokine axis, with the strength of network connections and module organization rearranged in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A) KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in the TSST-high and TSST-low groups. High-dose TSST-1 treatment was enriched in pathways related to interferon signaling, GPCR signaling, and potassium (K⁺) channel activity, while low-dose treatment primarily enriched pathways related to ion transport and neuronal processes. (B) Heatmaps of representative DEGs. The TSST-high group showed marked upregulation of IFIT1/2/3, OASL, HK2, CA9, and KRT16, whereas the TSST-low group specifically upregulated CXCL3, PPP1R14A, NECTIN4, TXNIP, and G0S2. (C) The lactate/pyruvate ratio in culture supernatants elevated with increasing TSST-1 concentration at both 24 h and 48 h, exhibiting a notable peak at 1000 ng/mL.\u003c/p\u003e \u003cp\u003eKEGG pathway analysis revealed that cells treated with high-dose TSST-1 showed significant enrichment of innate immune-related pathways, particularly interferon signaling, along with activation of GPCR signaling and potassium (K⁺) channel regulation pathways(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). In contrast, low-dose TSST-1 primarily affected ion transport, glutamate neurotransmitter release, and other neuronal signaling\u0026ndash;related pathways. Consistent with these pathway-level differences, we also compared the 20 genes exhibiting the most significant differential expression following TSST-1 treatment. Heatmap analysis of representative differentially expressed genes revealed that the transcriptional responses to high-dose TSST-1 and low-dose TSST-1 largely overlapped in glycolysis-related genes yet affected a distinct set of genes involved in cellular signalling and metabolic regulation (NDRG1, ENO2, HK2, LOX, SLC2A3, ARG2, CA9, ADSSL1, STC1). This suggests that the TSST-1's effects on epithelial cells are closely linked to glycolysis(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Consequently, we measured lactate and pyruvate levels in cells following TSST-1 treatment. The results demonstrated a dose-dependent increase in the lactate-to-pyruvate ratio at both 24 and 48 hours with increasing TSST-1 concentrations, with the most significant increase observed at 1000 ng/mL, thus providing further evidence for TSST-1\u0026ndash;induced metabolic reprogramming(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The elevated ratio reflects enhanced glycolytic flux under high-dose TSST-1 conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTSST toxins have been previously reported to impair cell viability and metabolic processes, including glycolysis and ion transport [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Consistent with these findings, our data demonstrate that TSST-1 markedly decreases the viability of human vaginal epithelial cells in both dose- and time-dependent manners. In accordance with the critical roles of the G₁ phase in RNA synthesis and protein accumulation, as well as the S phase in DNA replication [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], treatment of HVEC with TSST at varying concentrations significantly induces apoptosis, reduces cell viability, and inhibits proliferation through cell cycle disruption. In our investigation, flow cytometric analysis further revealed rapid increases in apoptotic populations following TSST-1 exposure, along with G₁-phase accumulation and decreased S-phase fractions, suggesting a reduced proliferative capacity. These observations align with prior findings on TSST-mediated cytotoxicity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], while expanding upon them by demonstrating a dose-graded pattern in vaginal epithelial cells, which has been inadequately addressed in previous research.\u003c/p\u003e \u003cp\u003eRNA-seq revealed significant enrichment of multiple pathways associated with immunological and metabolic stress under high-dose conditions, including interferon signaling, glycolysis, ion transport, and structural remodeling The prominence of glycolysis and ion transport genes suggests that cells were metabolically adapting to inflammatory stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], likely engaging compensatory pathways to regulate intracellular pH amidst lactate accumulation. These pathways typically attenuate proliferative drive while reinforcing checkpoint inhibition; therefore, the observed G1-phase arrest in flow cytometry can be interpreted as a functional manifestation of energy and metabolic stress at the transcriptional level. Inflammatory stimulation can drive a metabolic shift from oxidative phosphorylation to glycolysis\u0026mdash;a phenomenon known as the Warburg effect [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In alignment with this hypothesis, the observed increase in the lactate/pyruvate ratio in our work indicates that cells subjected to high TSST-1 undergo a Warburg-like shift, converting excess pyruvate to lactate rather than channeling it into mitochondrial oxidation. Meanwhile, mRNA expression of inflammatory cytokines, including IL-1β, IL-6, IL-8, MIP-α, TNF-α, and CXCL1 was markedly upregulated. For instance, IL-1β exposure has been shown to enhance glycolysis and increase lactate output in airway epithelial cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], while type I interferons can induce hexokinase 2 (HK2) and drive aerobic glycolysis in epithelial tissues. This demonstrates a distinct dose-dependent pattern: IL-1β and IL-6 peaked at moderate concentrations, whereas IL-8 and TNF-α continued to rise at higher doses. It is known that stimulation of genital tract epithelia induces the expression of antimicrobial peptides such as β-defensins and SLPI, which are regulated by pattern recognition receptors (PRRs) and hormonal signals, forming the epithelial effector arm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The combined phenotype of increased apoptosis, cell-cycle arrest, and cytokine upregulation aligns with transcriptomic data showing significant enrichment of pro-apoptotic and stress-related pathways, indicating that inflammatory activation is mechanistically linked to early epithelial injury.\u003c/p\u003e \u003cp\u003eAt the transcriptomic level, RNA-seq findings further corroborated and expanded upon the aforementioned phenotypic changes. Compared to the control group, low-dose TSST-1 treatment led to the upregulation of 898 genes and downregulation of 581 genes, whereas high-dose treatment resulted in only 726 upregulated and 378 downregulated genes. This suggests that moderate stimulation induces broader and more functional transcriptional reprogramming, whereas extremely high doses drive cells into a stress-suppressed state [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. GO/KEGG enrichment further demonstrated that low-dose responses were enriched in chemotaxis, antimicrobial defense, and receptor-ligand regulation, implying active immune recruitment, while high-dose treatment additionally triggered hypoxia-like metabolic stress and epithelial barrier remodeling. Although certain inflammatory cytokines were reduced in transcription within the cytokine\u0026ndash;receptor interaction pathway, their receptor/ligand counterparts remained at elevated levels, suggesting negative feedback regulation rather than reduced functional output. These findings indicate that TSST-1 does not trigger a linear inflammatory response but exhibits threshold-dependent and pathway-specific regulatory characteristics, representing a mechanistic refinement compared to previous works.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that TSST exposure can alter gene expression and trigger cellular stress and apoptosis, specifically through NF-κB-mediated production of TNF-α, IL-6, and IL-8 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our KEGG data similarly revealed activation of inflammatory and apoptotic pathways, including cytokine\u0026ndash;receptor interaction, chemokine signaling, TNF signaling, and stress-related networks such as HIF-1 (hypoxia) and glycolytic metabolism, supporting multiple converging axes of injury. HIF-1 enrichment implies reduced ATP production and suppression of the cell cycle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. IL-1β elevation further suggests IL-1 axis activation, consistent with reports showing IL-1β-mediated redistribution of ZO-1 and occludin, leading to epithelial barrier impairment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, IL-1β can disrupt epithelial junctions via the NF-κB/MLCK axis, enhancing permeability [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, our data together reinforce a mechanistic link between TSST-1, IL-1-driven inflammation, and barrier breakdown, which has not been systematically described previously in vaginal epithelial contexts.\u003c/p\u003e \u003cp\u003eWe also observed that the cellular effects of TSST-1 were not linearly cumulative across doses or timepoints but instead exhibited threshold and saturation behaviors, consistent with previous findings [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. IL-1β and IL-6 reached maximal induction at moderate doses, while IL-8 and TNF-α continued to increase, indicating separate activation thresholds and saturation dynamics. High toxin levels likely cause translation impairment, consistent with integrated stress response features in epithelial systems [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Reduced viability with preserved attachment during the first 24 h, followed by abrupt surges of apoptosis and detachment at 48\u0026ndash;72 h, indicates the presence of a damage threshold\u0026ndash;commitment point influenced by mitochondrial and checkpoint collapse [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Collectively, these results support a model in which TSST-1 not only activates inflammatory signaling but also simultaneously rewires cellular metabolism toward glycolysis to meet bioenergetic and biosynthetic demands\u0026mdash;a Warburg-like reprogramming that may sustain inflammation but predispose cells to injury through lactate accumulation and metabolic acidosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Finally, our study delineated the early epithelial responses to TSST-1 from phenotype to transcriptome. However, the immortalized VK2/E6E7 line lacks the complete mucosal barrier and immune microenvironment. Previous reports indicate that VK2/E6E7 cells cultured at an air\u0026ndash;liquid interface form multilayered, keratin-rich structures, with hormonal regulation significantly modulating barrier susceptibility [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, the observational time window of the present study was limited to 24\u0026ndash;72 h. Future studies should employ primary epithelial cells or organoid models and incorporate pathway inhibition experiments targeting inflammation-related receptor signaling pathways and their roles in regulating glycolytic metabolism, in order to further validate the underlying molecular mechanisms and assess long-term metabolic consequences.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study characterized the early epithelial responses to TSST-1 at both cellular and molecular levels, uncovering a coordinated pattern of inflammatory amplification, cell-cycle arrest, apoptosis, and barrier perturbation. These findings enhance our understanding of the initial pathophysiological processes underlying TSS, particularly regarding the role of mucosal epithelia as the \u0026ldquo;frontline\u0026rdquo; linking local exposure to systemic inflammation. Our findings lay a theoretical foundation for evaluating the risk of TSS induced by vaginal TSST-1 exposure and offer practical directions for early intervention. Efforts may focus on developing molecular or antibody-based agents that directly neutralize TSST-1 and testing their protective efficacy in epithelial models; conversely, targeting the epithelial IL-1/NF-κB axes with pharmacologic or biologic modulators could mitigate toxin-induced cellular damage, offering a rational basis for early prevention and therapeutic strategies against TSS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was partly funded by grants from the National Natural Science Fund of China (Grant No. 82150410452) and the Key Support Plan for Foreign Experts (Grant No. wgxz2022084L) to S.C.O.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eS.C.O. conceptualized the original research hypothesis, designed the project, and supervised this study. X.L. conducted the experiments, analyzed data, and drafted the manuscript. Y.Y. contributed to the methodology, data analysis, and project overview. C.K. provided assistance with resources, methods, and conceptual development. C.S. and H.Y. assisted with some methodology demonstration and provided analytical platforms. All authors reviewed the manuscript and approved the final version.\u003c/p\u003e\n\u003cp\u003eDeclaration of Competing Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing data generated and analysed during the current study have been deposited in the NCBI BioProject database under accession number \u003cstrong\u003ePRJNA1424937\u003c/strong\u003e. Other data supporting the findings of this study are included within the article and its supplementary materials.\u003c/p\u003e\u003cp\u003e\u0026ldquo;Ethics, Consent to Participate, and Consent to Publish declarations: not applicable.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Clinical trial number: not applicable.\u0026rdquo;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHe Y et al. Transcriptome Analysis of Caco-2 Cells upon the Exposure of Mycotoxin Deoxynivalenol and Its Acetylated Derivatives. Toxins (Basel), 2021. 13(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShepherd FR, et al. The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12\u0026thinsp;\u0026ndash;\u0026thinsp;4(+) memory T cells. Commun Biol. 2023;6(1):78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkins AL, et al. Toxic shock syndrome - the seven Rs of management and treatment. 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Int Immunol. 2021;33(3):127\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDufresne K, et al. Glucose Mediates Niche-Specific Repression of Staphylococcus aureus Toxic Shock Syndrome Toxin-1 through the Activity of CcpA in the Vaginal Environment. J Bacteriol. 2022;204(10):e0026922.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillon A, et al. Association of characteristics of tampon use with menstrual toxic shock syndrome in France. EClinicalMedicine. 2020;21:100308.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlievert PM, Davis CC. Device-Associated Menstrual Toxic Shock Syndrome. Clin Microbiol Rev, 2020. 33(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHajjeh RA, et al. Toxic shock syndrome in the United States: surveillance update, 1979 1996. Emerg Infect Dis. 1999;5(6):807\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Z, et al. Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4\u0026thinsp;+\u0026thinsp;T cells. Sci Rep. 2019;9(1):1864.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmitz M, et al. Streptococcal toxic shock syndrome in the intensive care unit. Ann Intensive Care. 2018;8(1):88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDufresne K, et al. TSST-1 promotes colonization of Staphylococcus aureus within the vaginal tract by activation of CD8(+) T cells. Infect Immun. 2025;93(2):e0043924.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu K, et al. Transcriptomics and flow cytometry reveals the cytotoxicity of aflatoxin B(1) and aflatoxin M(1) in bovine mammary epithelial cells. Ecotoxicol Environ Saf. 2021;209:111823.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtchade E et al. Toxic Shock Syndrome: A Literature Review. Antibiot (Basel), 2024. 13(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlievert PM et al. Staphylococcal Superantigens Stimulate Epithelial Cells through CD40 To Produce Chemokines. mBio, 2019. 10(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaefers MM, et al. Epithelial proinflammatory response and curcumin-mediated protection from staphylococcal toxic shock syndrome toxin-1. PLoS ONE. 2012;7(3):e32813.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIm J, et al. Gene expression profiling of bovine mammary gland epithelial cells stimulated with lipoteichoic acid plus peptidoglycan from Staphylococcus aureus. Int Immunopharmacol. 2014;21(1):231\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDufresne K, et al. Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production. J Biol Chem. 2024;300(7):107455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee PK, et al. Effects of staphylococcal toxic shock syndrome toxin 1 on aortic endothelial cells. J Infect Dis. 1991;164(4):711\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertoli C, Skotheim JM, de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol. 2013;14(8):518\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto-Heredero G, et al. Glycolysis - a key player in the inflammatory response. FEBS J. 2020;287(16):3350\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDas S, Jayaratne R, Barrett KE. The Role of Ion Transporters in the Pathophysiology of Infectious Diarrhea. Cell Mol Gastroenterol Hepatol. 2018;6(1):33\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManosalva C, et al. Corrigendum: Role of lactate in inflammatory processes: friend or foe. Front Immunol. 2025;16:1553925.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian X, et al. IL-1/inhibitory kappaB kinase epsilon-induced glycolysis augment epithelial effector function and promote allergic airways disease. J Allergy Clin Immunol. 2018;142(2):435\u0026ndash;e45010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarbrough VL, Winkle S, Herbst-Kralovetz MM. Antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications. Hum Reprod Update. 2015;21(3):353\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerrick BA, et al. RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats. PLoS ONE. 2013;8(4):e61768.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, et al. NF-kappaB signaling in inflammation. Signal Transduct Target Ther. 2017;2:17023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimura K, Teranishi S, Nishida T. Interleukin-1beta-induced disruption of barrier function in cultured human corneal epithelial cells. Invest Ophthalmol Vis Sci. 2009;50(2):597\u0026ndash;603.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaminsky LW, Al-Sadi R, Ma TY. IL-1beta and the Intestinal Epithelial Tight Junction Barrier. Front Immunol. 2021;12:767456.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight junction permeability. J Immunol. 2007;178(7):4641\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimber I, et al. Toxic shock syndrome: characterization of human immune responses to TSST-1 and evidence for sensitivity thresholds. Toxicol Sci. 2013;134(1):49\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaulkner L, et al. The mechanism of superantigen-mediated toxic shock: not a simple Th1 cytokine storm. J Immunol. 2005;175(10):6870\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabon JJ, Varghese LN, Nicola NA. Inhibition of IL-6 family cytokines by SOCS3. Semin Immunol. 2014;26(1):13\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X, et al. Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells. BMC Cell Biol. 2007;8:17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiesenberg BP, et al. Stress-Mediated Attenuation of Translation Undermines T-cell Activity in Cancer. Cancer Res. 2022;82(23):4386\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta S, et al. The mitochondrial death pathway: a promising therapeutic target in diseases. J Cell Mol Med. 2009;13(6):1004\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarreno-Florez GP, et al. Interferon signaling drives epithelial metabolic reprogramming to promote secondary bacterial infection. PLoS Pathog. 2023;19(11):e1011719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee Y et al. Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface. Viruses, 2016. 8(9).\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TSST-1, human vaginal epithelial cells, cytotoxicity, apoptosis, cell-cycle, inflammatory factors, RNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-8800664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8800664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eToxic shock syndrome toxin-1 (TSST-1) is a critical superantigen produced by certain staphylococcal strains, closely associated with menstrual toxic shock syndrome and mucosal inflammation. While the systemic superantigenic function of TSST-1 is well-established, its early cytotoxic and inflammatory effects on human vaginal epithelial cells (HVEC) remain poorly characterized. This study, therefore, seeks to investigate the initial temporal dynamics of TSST-1-induced cell viability, inflammatory cytokine responses, and barrier disruption in a model of human vaginal epithelium, along with a detailed analysis of the transcriptome.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe evaluated the impact of recombinant TSST-1 on HVEC at different doses and treatment durations. Subsequent exposure assessments included cell viability, apoptosis, and cell cycle distribution via flow cytometry, alongside inflammatory gene expression and transcriptomic profiling.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTSST-1 could significantly reduce cell viability and proliferation in a dose- and time-dependent manner, accompanied by increased apoptosis and marked G1 phase arrest. In contrast, early exposure did not result in a uniform upregulation of all inflammatory mediators; rather, multiple cytokines exhibited dose-dependent segmented response patterns instead of a linear increase. RNA sequencing analysis of samples treated with 125 or 1000 ng/mL TSST-1 for 48 hours revealed extensive transcriptional reprogramming involving chemokine signaling, cytokine-receptor interactions, epithelial stress-related pathways, and metabolic rewiring. Notably, the number of differentially expressed genes induced by moderate-dose TSST-1 exceeded that of high-dose treatment, while high-dose TSST-1 specifically enriched pathways related to hypoxia and glycolysis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings demonstrate that TSST-1 can trigger cytotoxicity, apoptosis, cell cycle disruption, and a graded inflammatory response in vaginal epithelial cells, providing a systematic transcriptomic framework for understanding the early mucosal responses induced by TSST-1.\u003c/p\u003e","manuscriptTitle":"Transcriptomic Profiling of TSST-1–Induced Cytotoxicity and Inflammatory Responses in Human Vaginal Epithelial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 00:29:35","doi":"10.21203/rs.3.rs-8800664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T07:48:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T14:48:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T19:17:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296226080668048071336867957197503171576","date":"2026-04-30T17:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184963616283956977690343209959647251390","date":"2026-04-07T07:31:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130662544491401333352621290431668569512","date":"2026-04-06T21:19:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"97453668943693689323955185854775403024","date":"2026-04-05T20:50:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108204423265273165813873320280612015625","date":"2026-04-03T08:55:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-03T04:02:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T12:13:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-19T11:19:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-02-19T11:14:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6662c935-38c0-4d95-9be5-f9f46bbbf25a","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T07:48:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T14:48:43+00:00","index":96,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T19:17:20+00:00","index":84,"fulltext":""},{"type":"reviewerAgreed","content":"296226080668048071336867957197503171576","date":"2026-04-30T17:49:20+00:00","index":83,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T07:54:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 00:29:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8800664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8800664","identity":"rs-8800664","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}