Toxic Shock Syndrome (TSS) Caused by Group A Streptococcus: Novel Insights Within the Context of a Familiar Clinical Syndrome.

The emergence of invasive infections attributed to group A Streptococcus (GAS) infections, has resurged since the 1980s. The recent surge in reports of toxic shock syndrome due to GAS in Japan in 2024, while sensationalized in the media, does not represent a novel infectious disease per se, as its diagnosis, treatment, and prevention are already well-established. However, due to signs of increasing incidence since 2011, further research is needed. Health authorities in neighboring countries like The Republic of Korea should not only issue travel advisories but also establish meticulous surveillance systems and initiate epidemiological studies on the genotypic variations of this disease while awaiting various epidemiological research findings from Japan.

Feasibility of using a combination of staphylococcal superantigen-like proteins 3, 7 and 11 in a fusion vaccine for Staphylococcus aureus.

Staphylococcus aureus is a significant bacterial pathogen in both community and hospital settings, and the escalation of antimicrobial-resistant strains is of immense global concern. Vaccination is an inviting long-term strategy to curb staphylococcal disease, but identification of an effective vaccine has proved to be challenging. Three well-characterized, ubiquitous, secreted immune evasion factors from the staphylococcal superantigen-like (SSL) protein family were selected for the development of a vaccine. Wild-type SSL3, 7 and 11, which inhibit signaling through Toll-like receptor 2, cleavage of complement component 5 and neutrophil function, respectively, were successfully combined into a stable, active fusion protein (PolySSL7311). Vaccination of mice with an attenuated form of the PolySSL7311 protein stimulated significantly elevated specific immunoglobulin G and splenocyte proliferation responses to each component relative to adjuvant-only controls. Vaccination with PolySSL7311, but not a mixture of the individual proteins, led to a > 102 reduction in S. aureus tissue burden compared with controls after peritoneal challenge. Comparable antibody responses were elicited after coadministration of the vaccine in either AddaVax (an analog of MF59) or an Alum-based adjuvant; but only AddaVax conferred a significant reduction in bacterial load, aligning with other studies that suggest both cellular and humoral immune responses are necessary for protective immunity to S. aureus. Anti-sera from mice immunized with PolySSL7311, but not individual proteins, partially neutralized the functional activities of SSL7. This study confirms the importance of these SSLs for the survival of S. aureus in vivo and suggests that PolySSL7311 is a promising vaccine candidate.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

The Celiac-Disease Superantigen Oligomerizes and Increases Permeability in an Enterocyte Cell Model.

Celiac disease (CeD) is an autoimmune disorder triggered by gluten proteins, affecting approximately 1 % of the global population. The 33-mer deamidated gliadin peptide (DGP) is a metabolically modified wheat-gluten superantigen for CeD. Here, we demonstrate that the 33-mer DGP spontaneously assembles into oligomers with a diameter of approximately 24 nm. The 33-mer DGP oligomers present two main secondary structural motifs-a major polyproline II helix and a minor ß-sheet structure. Importantly, in the presence of 33-mer DGP oligomers, there is a statistically significant increase in the permeability in the gut epithelial cell model Caco-2, accompanied by the redistribution of zonula occludens-1, a master tight junction protein. These findings provide novel molecular and supramolecular insights into the impact of 33-mer DGP in CeD and highlight the relevance of gliadin peptide oligomerization.

Staphylococcal superantigen-like protein 3 triggers murine mast cell adhesion by binding to CD43 and augments mast cell activation.

Staphylococcus aureus is a noteworthy pathogen in allergic diseases, as four staphylococcal exotoxins activate mast cells, a significant contributor to inflammation, in an IgE-independent manner. Although the adhesion of mast cells is an essential process for their immune responses, only a small number of exotoxins have been reported to affect the process. Here, we demonstrated that staphylococcal superantigen-like (SSL) 3, previously identified as a toll-like receptor 2 agonist, induced the adhesion of murine bone marrow-derived mast cells to culture substratum. SSL3-induced adhesion was mediated by fibronectin in an Arg-Gly-Asp (RGD) sequence-dependent manner, suggesting the integrins were involved in the process. Additionally, SSL3 was found to bind to an anti-adhesive surface protein CD43. SSL3 induced the adhesion of HEK293 cells expressing exogenous CD43, suggesting that CD43 is the target molecule for adhesion induced by SSL3. Evaluation of SSL3-derived mutants showed that the C-terminal region (253-326), specifically T285 and H307, are necessary to induce adhesion. SSL3 augmented the IL-13 production of mast cells in response to immunocomplex and SSL12. These findings reveal a novel function of SSL3, triggering cell adhesion and enhancing mast cell activation. This study would clarify the correlation between S. aureus and allergic diseases such as atopic dermatitis.

Evolutionary dynamics of the novel ST22-PT methicillin-resistant Staphylococcus aureus clone co-harbouring Panton-Valentine leucocidin and duplicated toxic shock syndrome toxin 1 genes.

OBJECTIVES: Globally, the isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) harbouring both the Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) genes is rare. However, we encountered an outbreak of the ST22-PT clone exhibiting this phenotype in Japan. Notably, the TSST-1 gene was duplicated in most of the strains. This study aimed to elucidate the mechanisms underlying this gene duplication. METHODS: A total of 90 MRSA isolates were collected from the skin of outpatients in Fukuoka City, Japan, between 2017 and 2019. Whole-genome sequencing was performed on MRSA strains that were PVL and TSST-1 positive. RESULTS: A total of 43 (47.8%) strains produced TSST-1, 20 (22.2%) produced PVL, and 16 (17.8%) produced both. Fifteen isolates were classified as ST22/SCCmec type IVa (ST22-PT clone) and one as ST1/SCCmec type V (ST1-PT clone). Three distinct ST22-PT clones were identified: Fukuoka clone I (one PVL gene and one TSST-1 gene), Fukuoka clone II (addition of a TSST-1 gene to Fukuoka clone I), and Fukuoka clone III (marked by a chromosomal inversion in a large region from Fukuoka clone II). DISCUSSION: Fukuoka clone I may have integrated a novel pathogenicity island bearing the TSST-1 gene, leading to the emergence of Fukuoka clone II with a duplicated TSST-1 gene. This duplication subsequently instigated a chromosomal inversion in a large region owing to the homologous sequence surrounding TSST-1, giving rise to Fukuoka clone III. These findings provide crucial insights into the genetic evolution of MRSA.

Vaginal community state types (CSTs) alter environmental cues and production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1).

Menstrual toxic shock syndrome (mTSS) is a rare but life-threatening disease associated with the use of high-absorbency tampons. The production of the Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) superantigen is involved in nearly all cases of mTSS and is tightly controlled by regulators responding to the environment. In the prototypic mTSS strain S. aureus MN8, the major repressor of TSST-1 is the carbon catabolite protein A (CcpA), which responds to glucose concentrations in the vaginal tract. Healthy vaginal Lactobacillus species also depend on glucose for both growth and acidification of the vaginal environment through lactic acid production. We hypothesized that interactions between the vaginal microbiota [herein referred to as community state types (CSTs)] and S. aureus MN8 depend on environmental cues and that these interactions subsequently affect TSST-1 production. Using S. aureus MN8 ΔccpA growing in various glucose concentrations, we demonstrate that the supernatants from different CSTs grown in vaginally defined medium (VDM) could significantly decrease tst expression. When co-culturing CST species with MN8 ∆ccpA, we show that Lactobacillus jensenii completely inhibits TSST-1 production in conditions mimicking healthy menstruation or mTSS. Finally, we show that growing S. aureus in "unhealthy" or "transitional" CST supernatants results in higher interleukin 2 (IL-2) production from T cells. These findings suggest that dysbiotic CSTs may encourage TSST-1 production in the vaginal tract and further indicate that the CSTs are likely important for the protection from mTSS.IMPORTANCEIn this study, we investigate the impact of the vaginal microbiota against Staphylococcus aureus in conditions mimicking the vaginal environment at various stages of the menstrual cycle. We demonstrate that Lactobacillus jensenii can inhibit toxic shock syndrome toxin-1 (TSST-1) production, suggesting the potential for probiotic activity in treating and preventing menstrual toxic shock syndrome (mTSS). On the other side of the spectrum, "unhealthy" or "transient" bacteria such as Gardnerella vaginalis and Lactobacillus iners support more TSST-1 production by S. aureus, suggesting that community state types are important in the development of mTSS. This study sets forward a model for examining contact-independent interactions between pathogenic bacteria and the vaginal microbiota. It also demonstrates the necessity of replicating the environment when studying one as dynamic as the vagina.

Interplay between Staphylococcus aureus and the vaginal microbiota.

Staphylococcus aureus is a proficient colonizer and opportunistic pathogen which can lead to vaginal dysbiosis, aerobic vaginitis, or life-threatening menstrual toxic shock syndrome. Here we explore the complex but underappreciated interactions that S. aureus may impose on the vaginal environment leading to additional disease outcomes.

Novel insights into the immune response to bacterial T cell superantigens.

Bacterial T cell superantigens (SAgs) are a family of microbial exotoxins that function to activate large numbers of T cells simultaneously. SAgs activate T cells by direct binding and crosslinking of the lateral regions of MHC class II molecules on antigen-presenting cells with T cell receptors (TCRs) on T cells; these interactions alter the normal TCR-peptide-MHC class II architecture to activate T cells in a manner that is independent of the antigen specificity of the TCR. SAgs have well-recognized, central roles in human diseases such as toxic shock syndrome and scarlet fever through their quantitative effects on the T cell response; in addition, numerous other consequences of SAg-driven T cell activation are now being recognized, including direct roles in the pathogenesis of endocarditis, bloodstream infections, skin disease and pharyngitis. In this Review, we summarize the expanding family of bacterial SAgs and how these toxins can engage highly diverse adaptive immune receptors. We highlight recent findings regarding how SAg-driven manipulation of the adaptive immune response may operate in multiple human diseases, as well as contributing to the biology and life cycle of SAg-producing bacterial pathogens.

Blocking Superantigen-Mediated Diseases: Challenges and Future Trends.

Superantigens are virulence factors secreted by microorganisms that can cause various immune diseases, such as overactivating the immune system, resulting in cytokine storms, rheumatoid arthritis, and multiple sclerosis. Some studies have demonstrated that superantigens do not require intracellular processing and instated bind as intact proteins to the antigen-binding groove of major histocompatibility complex II on antigen-presenting cells, resulting in the activation of T cells with different T-cell receptor Vß and subsequent overstimulation. To combat superantigen-mediated diseases, researchers have employed different approaches, such as antibodies and simulated peptides. However, due to the complex nature of superantigens, these approaches have not been entirely successful in achieving optimal therapeutic outcomes. CD28 interacts with members of the B7 molecule family to activate T cells. Its mimicking peptide has been suggested as a potential candidate to block superantigens, but it can lead to reduced T-cell activity while increasing the host's infection risk. Thus, this review focuses on the use of drug delivery methods to accurately target and block superantigens, while reducing the adverse effects associated with CD28 mimic peptides. We believe that this method has the potential to provide an effective and safe therapeutic strategy for superantigen-mediated diseases.

Epidemiological changes in invasive Streptococcus pyogenes infection during the UK alert period: A molecular comparative analysis from a tertiary Spanish hospital in 2023.

OBJECTIVES: To study the genomic epidemiology of Streptococcus pyogenes causing bloodstream infections (GAS-BSI) in a Spanish tertiary hospital during the United Kingdom invasive S. pyogenes outbreak alert. METHODS: Retrospective epidemiological analysis of GAS-BSI during the January-May 2017-2023 period. WGS was performed using Ion torrent GeneStudio™ S5 system for emm typing and identification of superantigen genes in S. pyogenes isolated during the 2022-2023 UK outbreak alert. RESULTS: During 2023, there were more cases of GAS-BSI compared to the same period of previous year with a non-significant increase in children. Fourteen isolates were sequenced. The emm1 (6/14, 42.9%) and emm12 (2/14, 14.3%) types predominated; 5 of 6 (75%) emm1 isolates were from the M1UK clone. The most detected superantigen genes were speG (12/14, 85.7%), speC (10/14, 71.4%), speJ (7/14, 50%), and speA (5/15, 33.3%). speA and speJ were predominant in M1UK clone. CONCLUSIONS: Our genomic epidemiology in 2023 is similar to the reported data from the UK outbreak alert in the same period and different from previous national S. pyogenes surveillance reports.

Putative staphylococcal enterotoxin possesses two common structural motifs for MHC-II binding.

Staphylococcus aureus has become a significant cause of health risks in humankind. Staphylococcal superantigens (SAgs) or enterotoxins are the key virulent factors that can exhibit acute diseases to severe life-threatening conditions. Recent literature reports S. aureus has steadily gained new enterotoxin genes over the past few decades. In spite of current knowledge of the established SAgs, several questions on putative enterotoxins are still remaining unanswered. Keeping that in mind, this study sheds light on a putative enterotoxin SEl26 to characterize its structural and functional properties. In-silico analyses indicate its close relation with the conventional SAgs, especially the zinc-binding SAgs. Additionally, important residues that are vital for the T-cell receptor (TcR) and major histocompatibility complex class II (MHC-II) interaction were predicted and compared with established SAgs. Besides, our biochemical analyses exhibited the binding of this putative enterotoxin with MHC-II, followed by regulating pro-inflammatory and anti-inflammatory cytokines.

Human Skin T Cells Express Conserved T-Cell Receptors that Cross-React with Staphylococcal Superantigens and CD1a.

Human Langerhans cells highly express CD1a antigen-presenting molecules. To understand the functions of CD1a in human skin, we used CD1a tetramers to capture T cells and determine their effector functions and TCR patterns. Skin T cells from all donors showed CD1a tetramer staining, which in three cases exceeded 10% of skin T cells. CD1a tetramer-positive T cells produced diverse cytokines, including IL-2, IL-4, IL-5, IL-9, IL-17, IL-22, and IFN-γ. Conserved TCRs often recognize nonpolymorphic antigen-presenting molecules, but no TCR motifs are known for CD1a. We detected highly conserved TCRs that used TRAV34 and TRBV28 variable genes, which is a known motif for recognition of staphylococcal enterotoxin B, a superantigen associated with atopic dermatitis. We found that these conserved TCRs did not respond to superantigen presented by CD1a, but instead showed a cross-reactive response with two targets: CD1a and staphylococcal enterotoxin B presented by classical major histocompatibility complex II. These studies identify a conserved human TCR motif for CD1a-reactive T cells. Furthermore, the demonstrated cross-reaction of T cells with two common skin-specific stimuli suggests a candidate mechanism by which CD1a and skin flora could synergize during natural immune response and in Staphylococcus-associated skin diseases.

Superantigen-fused T cell engagers for tumor antigen-mediated robust T cell activation and tumor cell killing.

Inadequate T cell activation has severely limited the success of T cell engager (TCE) therapy, especially in solid tumors. Enhancing T cell activity while maintaining the tumor specificity of TCEs is the key to improving their clinical efficacy. However, currently, there needs to be more effective strategies in clinical practice. Here, we design novel superantigen-fused TCEs that display robust tumor antigen-mediated T cell activation effects. These innovative drugs are not only armed with the powerful T cell activation ability of superantigens but also retain the dependence of TCEs on tumor antigens, realizing the ingenious combination of the advantages of two existing drugs. Superantigen-fused TCEs have been preliminarily proven to have good (>30-fold more potent) and specific (>25-fold more potent) antitumor activity in vitro and in vivo. Surprisingly, they can also induce the activation of T cell chemotaxis signals, which may promote T cell infiltration and further provide an additional guarantee for improving TCE efficacy in solid tumors. Overall, this proof-of-concept provides a potential strategy for improving the clinical efficacy of TCEs.

Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

Molecular Genetic Characterization of Streptococcus pyogenes Strains Isolated from Patients with Various Manifestations of Streptococcal Infection.

In 82 clinical strains of Streptococcus pyogenes (group A streptococci) isolated from patients with various manifestations of streptococcal infection, emm-typing revealed 27 emm-types (n=77) with a predominance of emm-89 (n=15; 18%), emm-75 (n=9; 11%), and emm-1 (n=6; 7%); types emm-3, emm-12, and emm-58 (n=4; 5% each) were found with almost equal frequency; other types were less common. The superantigen genes speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and SSA were identified in S. pyogenes strains using multiprimer PCR; the genes of the superantigen SpeA and cysteine proteinase SpeB were detected using real-time PCR. All the studied S. pyogenes strains contained superantigen genes, and 98% of the strains had several (from 2 to 7) genes. The number of variants of these sets reached 37; 2% of the strains contained only one superantigen gene. The distribution frequencies of superantigen genes in the studied strains were: speA - 43%; speC - 38%; speG - 93%; speH - 13%; speI - 6%; speJ - 24%; speK - 13%; speL and speM - 11% each; smeZ - 98%; SSA - 15%. All studied S. pyogenes strains contained the speB gene. Our studies have demonstrated that the sets of superantigen genes of group A streptococci are characterized by pronounced diversity to some extent associated with emm-type.

Detection of superantigens in Streptococcus pyogenes isolates based on whole genome sequencing data.

Streptococcus pyogenes causes a variety of human diseases ranging from uncomplicated respiratory tract and skin infections to severe invasive diseases possibly involving toxic shock syndrome. Besides the emm gene-encoded M protein, important virulence factors are pyrogenic exotoxins, referred to as superantigens. The National Reference Laboratory for Streptococcal Infections has newly introduced bioinformatics tools for processing S. pyogenes whole genome sequencing data. Using the SRST2 software and BV-BRC platform, WGS data of 10 S. pyogenes isolates from patients with invasive disease were analysed, and emm type, sequence type, and superantigen encoding gene profiles were determined. The Unicycler assembly pipeline with the SPAdes de novo assembler was used to assemble genome sequences from short reads.

Toxin exposure and HLA alleles determine serum antibody binding to toxic shock syndrome toxin 1 (TSST-1) of Staphylococcus aureus.

Life-threatening toxic shock syndrome is often caused by the superantigen toxic shock syndrome toxin-1 (TSST-1) produced by Staphylococcus aureus. A well-known risk factor is the lack of neutralizing antibodies. To identify determinants of the anti-TSST-1 antibody response, we examined 976 participants of the German population-based epidemiological Study of Health in Pomerania (SHIP-TREND-0). We measured anti-TSST-1 antibody levels, analyzed the colonization with TSST-1-encoding S. aureus strains, and performed a genome-wide association analysis of genetic risk factors. TSST-1-specific serum IgG levels varied over a range of 4.2 logs and were elevated by a factor of 12.3 upon nasal colonization with TSST-1-encoding S. aureus. Moreover, the anti-TSST-1 antibody levels were strongly associated with HLA class II gene loci. HLA-DRB1*03:01 and HLA-DQB1*02:01 were positively, and HLA-DRB1*01:01 as well as HLA-DQB1*05:01 negatively associated with the anti-TSST-1 antibody levels. Thus, both toxin exposure and HLA alleles affect the human antibody response to TSST-1.

Stability and emetic activity of enterotoxin like X (SElX) with high carrier rate of food poisoning Staphylococcus aureus.

In order to analyze and clarify the thermal stability of food poisoning Staphylococcus aureus (S. aureus) enterotoxin-like X (SElX) and the biological characteristics of digestive enzymes, and to evaluate the risk of S. aureus carrying selx gene in food poisoning, the selx gene carrying rates of 165 strains isolated from 95 food poisoning events from 2006 to 2019 were first statistically analyzed. Subsequently, the purified recombinant SElX protein was digested and heated, and the superantigen activity was verified with mouse spleen cells and peripheral blood mononuclear cells of kittens. At the same time, the emetic activity and toxicity of SElX were evaluated using the kitten vomiting animal model, mice toxin model and in vitro cell models. The results showed the selx gene carrying rate of 165 food poisoning S. aureus strains was 90.30 %. SElX had significant resistance to heat treatment and pepsin digestion (pH = 4.0 and pH = 4.5), and had good superantigen activity and emetic activity. However, there is no significant lethal effect on mice and no significant toxicity to cells. Importantly, we found that SElX had an inhibitory effect on acidic mucus of goblet cells in various segments of the small intestine. The present study investigated the stability of SElX, and confirmed the emetic activity of SElX by establishing a kitten vomiting model for the first time, suggesting that SElX is a high risk toxin of food poisoning, which will provide new ideas for the prevention and control of S. aureus food poisoning.

Staphylococcal Enterotoxin C2 Mutant-Induced Antitumor Immune Response Is Controlled by CDC42/MLC2-Mediated Tumor Cell Stiffness.

As a biological macromolecule, the superantigen staphylococcal enterotoxin C2 (SEC2) is one of the most potent known T-cell activators, and it induces massive cytotoxic granule production. With this property, SEC2 and its mutants are widely regarded as immunomodulating agents for cancer therapy. In a previous study, we constructed an MHC-II-independent mutant of SEC2, named ST-4, which exhibits enhanced immunocyte stimulation and antitumor activity. However, tumor cells have different degrees of sensitivity to SEC2/ST-4. The mechanisms of immune resistance to SEs in cancer cells have not been investigated. Herein, we show that ST-4 could activate more powerful human lymphocyte granule-based cytotoxicity than SEC2. The results of RNA-seq and atomic force microscopy (AFM) analysis showed that, compared with SKOV3 cells, the softer ES-2 cells could escape from SEC2/ST-4-induced cytotoxic T-cell-mediated apoptosis by regulating cell softness through the CDC42/MLC2 pathway. Conversely, after enhancing the stiffness of cancer cells by a nonmuscle myosin-II-specific inhibitor, SEC2/ST-4 exhibited a significant antitumor effect against ES-2 cells by promoting perforin-dependent apoptosis and the S-phase arrest. Taken together, these data suggest that cell stiffness could be a key factor of resistance to SEs in ovarian cancer, and our findings may provide new insight for SE-based tumor immunotherapy.