Double-Enhanced Photothermal Lateral Flow Biosensor Based on Dual Gold Nanoparticle Conjugates.

Bacterial toxins emerge as the primary triggers of foodborne illnesses, posing a significant threat to human health. To ensure food safety, it is imperative to implement point-of-care testing methods. Lateral flow biosensors (LFBs) based on gold nanoparticles (GNPs) have been commonly used for rapid detection, but their applicationis limited by low sensitivity. Based on the localized surface plasmon resonance and photothermal effect of dual gold nanoparticle conjugates (DGNPs), we developed a smartphone-integrated photothermal LFB (PLFB) with double-enhanced colorimetric and photothermal sensitivity. Through numerical simulations, we verified that DGNPs have significantly enhanced photothermal performance compared to single 15 nm GNPs (SGNPs), and applied DGNPs in PLFB for the detection of staphylococcus enterotoxin A (SEA). The colorimetric and photothermal limits of detection of DGNPs-based PLFB for SEA were 0.091 and 0.0038 ng mL-1, respectively. Compared with the colorimetric detection of the SGNPs-based LFB, the colorimetric detection sensitivity of the DGNPs-based PLFB was increased by 10.7 times, and the photothermal detection sensitivity was further improved by 255.3 times. Moreover, the PLFB exhibits robust reproducibility and exceptional specificity and is applicable for detecting SEA in milk samples. This smartphone-integrated PLFB based on DGNPs allows users to detect toxins simply, conveniently, and quickly and has huge application potential in the field of food safety.

Induction of neutralizing antibodies and mucosal IgA through intranasal immunization with the receptor binding domain of SARS-CoV-2 spike protein fused with the type IIb E. coli heat-labile enterotoxin A subunit.

The outbreak of SARS-CoV-2 infections had led to the COVID-19 pandemic which has a significant impact on global public health and the economy. The spike (S) protein of SARS-CoV-2 contains the receptor binding domain (RBD) which binds to human angiotensin-converting enzyme 2 receptor. Numerous RBD-based vaccines have been developed and recently focused on the induction of neutralizing antibodies against the immune evasive Omicron BQ.1.1 and XBB.1.5 subvariants. In this preclinical study, we reported the use of a direct fusion of the type IIb Escherichia coli heat-labile enterotoxin A subunit with SARS CoV-2 RBD protein (RBD-LTA) as an intranasal vaccine candidate. The results showed that intranasal immunization with the RBD-LTA fusion protein in BALB/c mice elicited potent neutralizing antibodies against the Wuhan-Hu-1 and several SARS-CoV-2 variants as well as the production of IgA antibodies in bronchoalveolar lavage fluids (BALFs). Furthermore, the heterologous RBD representing the same strains used in the bivalent mRNA vaccine were used as a second-dose RBD-LTA/RBD protein booster after bivalent mRNA vaccination. The results showed that the neutralizing antibody titers elicited by the intranasal bivalent RBD-LTA/RBD protein booster were similar to the intramuscular bivalent mRNA booster, but the RBD-specific IgA titers in sera and BALFs significantly increased. Overall, this preclinical study suggests that the RBD-LTA fusion protein could be a promising candidate as a mucosal booster COVID-19 vaccine.

Epigallocatechin Gallate Alleviates Staphylococcal Enterotoxin A-Induced Intestinal Barrier Damage by Regulating Gut Microbiota and Inhibiting the TLR4-NF-κB/MAPKs-NLRP3 Inflammatory Cascade.

Natural phytochemicals have attracted increasing attention because of their promising ability to tackle bacteriotoxin-induced public safety concerns. However, it is unclear how natural phytochemicals regulate the intestinal barrier dysfunction caused by bacteriotoxin, such as staphylococcal enterotoxin A (SEA). This study aims to illustrate the in vitro and in vivo protective mechanism of epigallocatechin gallate (EGCG) on SEA-triggered intestinal barrier damage and inflammation. Results show that EGCG alleviates intestinal barrier damage by effectively inhibiting SEA-induced intestinal permeability increase, tight junction protein and mucin loss, and intestinal cell apoptosis. EGCG also reduces intestinal inflammation by suppressing the TLR4-NF-κB/MAPKs-NLRP3 pathway. Importantly, EGCG reverses gut microbiota dysbiosis and short-chain fatty acid (SCFA) content decrease induced by SEA. It is worth noting that this study also detects the direct interaction between the phytochemical and virulence factors and finds that EGCG effectively not only inhibits the secretion of SEA but also binds with the secreted SEA to attenuate its toxicity. Taken together, EGCG mitigates SEA-induced intestinal barrier dysfunction via gut microbiota SCFA-mediated TLR4-NF-κB/MAPKs-NLRP3 inflammatory cascade inhibition. Overall, this research provides enlightening insight into the application of bacteriotoxin-targeting natural compounds in the field of food safety and human wellness.

Enterotoxins A and B produced by Staphylococcus aureus increase cell proliferation, invasion and cytarabine resistance in acute myeloid leukemia cell lines.

As in the case of cancer, the risk of infection increases when the host's immune system is not working properly. It has been shown that toxins produced by the bacteria responsible for bacterial infections can alter the properties of cancer cells as well as their sensitivity to chemotherapy agents. Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens in acute myeloid leukemia (AML) patients and it produces several virulence factors, including Staphylococcal enterotoxin A (SEA) and Staphylococcal enterotoxin B (SEB). Cytotoxicity, transwell migration, invasion assays, and various transcriptomic and gene set enrichment (GSE) analyses were used to determine how SEA and SEB alter cell proliferation, migration, invasion, and Cytarabine (Cyt) resistance in AML cell lines. The treatment of AML cell lines with SEA/SEB caused an increase in cell proliferation and Cyt resistance. Toxins enhanced the proclivity of cells to migrate and invade, with around 50% of cells in the presence of SEA and SEB. Transcriptomic and gene set enrichment analyses, and subsequent PCR validations showed dysregulation of immune related genes and genesets. Apparently, this allows AML cells to escape and survive the undesirable environment created by toxins, possibly via the ER stress signaling pathway. Therefore, SEA and SEB can significantly alter the characteristics of AML cancer cells and evaluation of alterations in responsible immune genes and pathways may be crucial for controlling the progression of cancer. In addition, our results suggest that there may be a strong interaction between the immune related pathways and the ER signaling pathway.

Staphylococcus aureus enterotoxin A- and B-specific IgE in chronic obstructive pulmonary disease.

Sensitization to Staphylococcus aureus enterotoxins A (SEA) and B (SEB) has been associated with asthma severity, exacerbations, and disease control. Our study aimed to investigate if there are differences in serum SEA-IgE and SEB-IgE levels between patients with chronic obstructive pulmonary disease (COPD), asthma, and controls, and to assess the association between SE sensitization and COPD clinical parameters and Th2 inflammation biomarkers in two well-defined COPD cohorts. Our findings suggest that COPD patients do not exhibit higher SEA and SEB sensitization compared to asthma patients and controls. However, in COPD patients, the presence of atopy and allergy is associated with positivity for SEA-IgE and SEB-IgE. Consequently, these allergens may aid in identifying atopic or allergic subgroups within the COPD population, but they are not directly associated with the diagnosis of COPD, elevated circulating blood eosinophils, or fractional exhaled nitric oxide (FENO) levels.

Regulation of Staphylococcal Enterotoxin-Induced Inflammation in Spleen Cells from Diabetic Mice by Polyphenols.

Patients with diabetes are known to be more susceptible to infections following the establishment of Staphylococcus aureus in their nasal passages and on their skin. The present study evaluated the effects of staphylococcal enterotoxin A (SEA) on the immune responses of spleen cells derived from diabetic mice, and examined the effects of polyphenols, catechins, and nobiletin on inflammation-related gene expression associated with the immune response. (-)-Epigallocatechin gallate (EGCG), possessing hydroxyl groups, interacted with SEA, whereas nobiletin, possessing methyl groups, did not interact with SEA. The exposure of spleen cells derived from diabetic mice to SEA enhanced the expression of interferon gamma, suppressor of cytokine signaling 1, signal transducer and activator of transcription 3, interferon-induced transmembrane protein 3, Janus kinase 2, and interferon regulatory factor 3, suggesting that SEA sensitivity is variable in the development of diabetes. Both EGCG and nobiletin changed the expression of genes related to SEA-induced inflammation in spleen cells, suggesting that they inhibit inflammation through different mechanisms. These results may lead to a better understanding of the SEA-induced inflammatory response during diabetogenesis, and the establishment of methods to control these effects with polyphenols.

A food poisoning caused by ST7 Staphylococcal aureus harboring sea gene in Hainan province, China.

ST7 Staphylococcus aureus is highly prevalent in humans, pigs, as well as food in China; however, staphylococcal food poisoning (SFP) caused by this ST type has rarely been reported. On May 13, 2017, an SFP outbreak caused by ST7 S. aureus strains occurred in two campuses of a kindergarten in Hainan Province, China. We investigated the genomic characteristics and phylogenetic analysis of ST7 SFP strains combined with the 91 ST7 food-borne strains from 12 provinces in China by performing whole-genome sequencing (WGS). There was clear phylogenetic clustering of seven SFP isolates. Six antibiotic genes including blaZ, ANT (4')-Ib, tetK, lnuA, norA, and lmrS were present in all SFP strains and also showed a higher prevalence rate in 91 food-borne strains. A multiple resistance plasmid pDC53285 was present in SFP strain DC53285. Among 27 enterotoxin genes, only sea and selx were found in all SFP strains. A ФSa3int prophage containing type A immune evasion cluster (sea, scn, sak, and chp) was identified in SFP strain. In conclusion, we concluded that this SFP event was caused by the contamination of cakes with ST7 S. aureus. This study indicated the potential risk of new emergencing ST7 clone for SFP.

Aggregation-Induced Red Emission Nanoparticle-Based Lateral Flow Immunoassay for Highly Sensitive Detection of Staphylococcal Enterotoxin A.

Staphylococcal enterotoxin A (SEA) has presented enormous difficulties in dairy food safety and the sensitive detection of SEA provides opportunities for effective food safety controls and staphylococcal food poisoning tracebacks. Herein, a novel aggregation-induced emission (AIE)-based sandwich lateral flow immunoassay (LFIA) was introduced to detect SEA by using red-emissive AIE nanoparticles (AIENPs) as the fluorescent nanoprobe. The nanoprobe was constructed by directly immobilising antibodies on boronate-tagged AIENPs (PBA-AIENPs) via a boronate affinity reaction, which exhibited a high SEA-specific affinity and remarkable fluorescent performance. Under optimal conditions, the ultrasensitive detection of SEA in pasteurised milk was achieved within 20 min with a limit of detection of 0.04 ng mL-1. The average recoveries of the PBA-AIENP-LFIA ranged from 91.3% to 117.6% and the coefficient of variation was below 15%. It was also demonstrated that the PBA-AIENP-LFIA had an excellent selectivity against other SE serotypes. Taking advantage of the excellent sensitivity of this approach, real chicken and salad samples were further analysed, with a high versatility and accuracy. The proposed PBA-AIENP-LFIA platform shows promise as a potent tool for the identification of additional compounds in food samples as well as an ideal test method for on-site detections.

Optimization and evaluation of a multiplex PCR assay for detection of Staphylococcus aureus and its major virulence genes for assessing food safety.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

Staphylococcal enterotoxin A induces DNA damage in hepatocytes and liver tissues.

Staphylococcal enterotoxin A (SEA) is a foodborne bacterial toxin that can cause food poisoning, but little research has been done on the DNA damage caused by SEA. The aim of this research was to investigate the action of SEA in inducing DNA damage and oxidative stress response in hepatocytes and liver tissues. After treating HL-7702 and BRL-3A cells with different concentrations of SEA (0, 300, 600 ng/mL and 0, 400, 800 ng/mL), the production of phosphorylated H2AX (γH2AX) and p53 binding protein 1 (53BP1) aggregates was detected by confocal fluorescence microscopy, and the increases in ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (Chk2), p53 protein expression were assessed by Western blot analysis, while increased reactive oxygen species (ROS) content was confirmed by flow cytometry and fluorescence probe. The genotoxicity of SEA to cells was attenuated after the addition of an oxidative inhibitor, demonstrating that SEA induced intracellular DNA damage through the oxidative pathway and a dose-dependent relationship was observed between the oxidation index and SEA. These experimental results deepen our understanding of SEA damage to cells at the genetic level, and provide a new orientation for the prevention and cure of food borne diseases caused by Staphylococcal enterotoxins (SEs).

Evaluation of the Antimicrobial Activity of ZnO Nanoparticles against Enterotoxigenic Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a Gram-positive bacteria considered one of the leading causes of community and hospital-acquired illnesses or public health concerns. Antibiotic resistance in this microorganism is one of the greatest issues in global health care. The use of metal nanoparticles and their oxides is one of the potential approaches to combating bacteria resistance to antibiotics. The antibacterial properties of ZnO NPs against enterotoxigenic S. aureus were studied. ZnO NPs were tested in vitro by agar diffusion test. They resulted in 26 and 22 mm zones of inhibition for a size of 20 nm and a concentration of 20 mM against 105 and 107 CFU/mL S. aureus, respectively. The MIC of ZnO NPs of various sizes, 20 and 50 nm, with 105 CFU/mL was 2.5 and 5 mM, respectively. MIC with 107 CFU/mL was five mM for 20 and 50 nm ZnO NPs. Further, the highest growth reduction percentage, 98.99% in the counts of S. aureus was achieved by ZnO NPs of size 20 nm and concentration of 10 mM. Moreover, the obtained ELISA results indicated a significantly decreased concentration of enterotoxin A with all concentrations and sizes of ZnO NPs. PCR analysis showed a significant effect on sea gene in response to ZnO NPs treatments leading to loss of the gene, unlike the unaffected nuc gene. Moreover, morphological changes and cell shape distortion were detected by scanning electron microscope for bacterial cells treated with ZnO NPs.

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis.

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.

Cytotoxic Efficiency of Human CD8+ T Cell Memory Subtypes.

Immunological memory is important to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are well defined, single TEM and TCM cell cytotoxicity has not been quantified. To quantify cytotoxic efficiency of TEM and TCM, we developed a FRET-based single cell fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis, secondary necrosis following apoptosis, and primary necrosis after TEM- or TCM-target cell contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic efficiency of TEM compared to TCM, as quantified by target cell apoptosis and secondary necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in TEM compared to TCM. Higher perforin levels (likely in combination with higher granzyme levels) mediate higher cytotoxic efficiency of TEM compared to TCM. Both, TEM and TCM need the same time to find their targets, however contact time between CTL and target, time to induce apoptosis, and time to induce secondary necrosis are all shorter for TEM. In addition, immune synapse formation in TEM appears to be slightly more efficient than in TCM. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanisms is important to optimize future subset-based immune therapies.

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk.

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.

Effects of Growth Stage on the Characterization of Enterotoxin A-Producing Staphylococcus aureus-Derived Membrane Vesicles.

Virulence factors, such as staphylococcal enterotoxin A (SEA), are contained within membrane vesicles (MVs) in the cell membrane of Staphylococcus aureus. In this study, the effects of the growth stage on quantitative and qualitative changes in the components contained in the MVs of S. aureus SEA-producing strains were examined. Changes in the expression levels of S. aureus genes were examined at each growth stage; phenol-soluble modulin (PSM) gene reached a maximum after 8 h, and the expression of cell membrane-related genes was decreased after 6 h. Based on these gene expression patterns, MVs were prepared at 6, 17, and 24 h. The particle size of MVs did not change depending on the growth stage. MVs prepared after culture for 17 h maintained their particle size when stored at 23 °C. The amount of SEA in the culture supernatant and MVs were not correlated. Bifunctional autolysin, a protein involved in cell wall biosynthesis/degradation, was increased in MVs at 17 h. The expression pattern of inflammation-related genes in human adult low calcium high temperature (HaCaT) cells induced by MVs was different for each growth stage. The inclusion components of S. aureus-derived MVs are selective, depend on the stage of growth, and may play an important role in toxicity.

Staphylococcal Enterotoxin A Induces Intestinal Barrier Dysfunction and Activates NLRP3 Inflammasome via NF-κB/MAPK Signaling Pathways in Mice.

Staphylococcal enterotoxin A (SEA), the toxin protein secreted by Staphylococcus aureus, can cause staphylococcal food poisoning outbreaks and seriously threaten global public health. However, little is known about the pathogenesis of SEA in staphylococcal foodborne diseases. In this study, the effect of SEA on intestinal barrier injury and NLRP3 inflammasome activation was investigated by exposing BALB/c mice to SEA with increasing doses and a potential toxic mechanism was elucidated. Our findings suggested that SEA exposure provoked villi injury and suppressed the expression of ZO-1 and occludin proteins, thereby inducing intestinal barrier dysfunction and small intestinal injury in mice. Concurrently, SEA significantly up-regulated the expression of NLRP3 inflammasome-associated proteins and triggered the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in jejunum tissues. Notably, selective inhibitors of MAPKs and NF-κB p65 ameliorated the activation of NLRP3 inflammasome stimulated by SEA, which further indicated that SEA could activate NLRP3 inflammasome through NF-κB/MAPK pathways. In summary, SEA was first confirmed to induce intestinal barrier dysfunction and activate NLRP3 inflammasome via NF-κB/MAPK signaling pathways. These findings will contribute to a more comprehensive understanding of the pathogenesis of SEA and related drug-screening for the treatment and prevention of bacteriotoxin-caused foodborne diseases via targeting specific pathways.

BCL11B Upregulates the Expression of RelA in T Cells Stimulated with Staphylococcal Enterotoxin A.

We explored the potential link between RelA and BCL11B transcription factors. To this end, Jurkat and Raji cells (Jurkat:Raji 10:1), as well as normal human peripheral blood T cells, were activated by staphylococcal enterotoxin A (SEA) and the expressions of both BCL11B and RelA mRNA and proteins were detected. BCL11B small interfering RNA was then transduced into Jurkat cells. Under the effect of SEA stimulation, the expression of BCL11B and RelA mRNA increased in two types of T cell lines over time, and the results were comparable with the levels of expression of BCL11B and RelA proteins. In the BCL11B-knockdown cells, the expression of RelA protein did not increase. These findings suggest that BCL11B regulates RelA expression in Jurkat cells and human peripheral blood T cells from healthy donors via the T-cell receptor signaling pathway.

Bisdemethoxycurcumin Reduces Methicillin-Resistant Staphylococcus aureus Expression of Virulence-Related Exoproteins and Inhibits the Biofilm Formation.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen of nosocomial infection, which is resistant to most antibiotics. Presently, anti-virulence therapy and anti-biofilm therapy are considered to be promising alternatives. In the current work, we investigated the influence of bisdemethoxycurcumin (BDMC) on the virulence-related exoproteins and the biofilm formation using a reference strain and clinic isolated strains. Western blotting, quantitative RT-PCR, and tumor necrosis factor (TNF) release assay were performed to assess the efficacy of BDMC in reducing the expression of Staphylococcus enterotoxin-related exoproteins (enterotoxin A, enterotoxin B) and α-toxin in MRSA. The anti-biofilm activity of BDMC was evaluated through a biofilm inhibition assay. The study suggests that sub-inhibitory concentrations of BDMC significantly inhibited the expression of sea, seb, and hla at the mRNA level in MRSA. Moreover, the expression of virulence-related exoproteins was significantly decreased by down-regulating accessory gene regulator agr, and the inhibition of biofilms formation was demonstrated by BDMC at sub-inhibitory concentrations. Consequently, the study suggests that BDMC may be a potential natural antibacterial agent to release the pressure brought by antibiotic resistance.

Immunoinformatics analysis and evaluation of recombinant chimeric triple antigen toxoid (r-HAB) against Staphylococcus aureus toxaemia in mouse model.

Staphylococcus aureus is a serious pathogen unleashing its virulence through several classes of exotoxins such as hemolysins and enterotoxins. In this study, we designed a novel multi-antigen subunit vaccine which can induce innate, humoral and cellular immune responses. Alpha hemolysin, enterotoxins A and B were selected as protective antigens for combining into a triple antigen chimeric protein (HAB). Immunoinformatics analysis predicted HAB protein as a suitable vaccine candidate for inducing both humoral and cellular immune responses. Tertiary structure of the HAB protein was predicted and validated through computational approaches. Docking studies were performed between the HAB protein and mice TLR2 receptor. Furthermore, we constructed and generated recombinant HAB (r-HAB) protein in E. coli and studied its toxicity, immunogenicity and protective efficacy in a mouse model. Triple antigen chimeric protein (r-HAB) was found to be highly immunogenic in mouse as the anti-r-HAB hyperimmune serum was strongly reactive to all three native exotoxins on Western blot. In vitro toxin neutralization assay using anti-r-HAB antibodies demonstrated > 75% neutralization of toxins on RAW 264.7 cell line. Active immunization with r-HAB toxoid gave ~ 83% protection against 2 × lethal dosage of secreted exotoxins. The protection was mediated by induction of strong antibody responses that neutralized the toxins. Passive immunization with anti-r-HAB antibodies gave ~ 50% protection from lethal challenge. In conclusion, in vitro and in vivo testing of r-HAB found the molecule to be nontoxic, highly immunogenic and induced excellent protection towards native toxins in actively immunized and partial protection to passively immunized mice groups. KEY POINTS: • HAB protein was computationally designed to induce humoral and cellular responses. • r-HAB protein was found to be nontoxic, immunogenic and protective in mouse model. • r-HAB conferred protection against lethal challenge in active and passive immunization.

Effect of (-)-Epigallocatechin Gallate on Activation of JAK/STAT Signaling Pathway by Staphylococcal Enterotoxin A.

Staphylococcal enterotoxin A (SEA), which is a superantigen toxin protein, binds to cytokine receptor gp130. Gp130 activates intracellular signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The effects of SEA on the JAK/STAT signaling pathway in mouse spleen cells were examined. After treatment with SEA, mRNA expression levels of interferon gamma (IFN-γ) and suppressor of cytokine-signaling 1 (SOCS1) increased. SEA-induced IFN-γ and SOCS1 expression were decreased by treatment with (-)-epigallocatechin gallate (EGCG). The phosphorylated STAT3, Tyr705, increased significantly in a SEA concentration-dependent manner in mouse spleen cells. Although (-)-3″-Me-EGCG did not inhibit SEA-induced phosphorylated STAT3, EGCG and (-)-4″-Me-EGCG significantly inhibited SEA-induced phosphorylated STAT3. It was thought that the hydroxyl group at position 3 of the galloyl group in the EGCG was responsible for binding to SEA and suppressing SEA-induced phosphorylation of STAT3. Through protein thermal shift assay in vitro, the binding of the gp130 receptor to SEA and the phosphorylation of STAT3 were inhibited by the interaction between EGCG and SEA. As far as we know, this is the first report to document that EGCG inhibits the binding of the gp130 receptor to SEA and the associated phosphorylation of STAT3.