Transcriptomic analysis of the antimicrobial activity of prodigiosin against Cutibacterium acnes.

Prodigiosin, a red pigment produced by Hahella chejuensis, a marine-derived microorganism, has several biological functions, including antimicrobial activity and inflammatory relief. In this study, the antibacterial activity of prodigiosin against skin microorganisms was explored. Paper disc assay on skin bacterial cells revealed that Cutibacterium acnes related to acne vulgaris highly susceptible to prodigiosin. MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) were determined on Cutibacterium species. The RNA-seq analysis of prodigiosin-treated C. acnes cells was performed to understand the antibacterial mechanism of prodigiosin. Among changes in the expression of hundreds of genes, the expression of a stress-responsive sigma factor encoded by sigB increased. Conversely, the gene expression of cell wall biosynthesis and energy metabolism was inhibited by prodigiosin. Specifically, the expression of genes related to the metabolism of porphyrin, a pro-inflammatory metabolite, was significantly reduced. Therefore, prodigiosin could be used to control C. acnes. Our study provided new insights into the antimicrobial mechanism of prodigiosin against C. acnes strains.

The Fatal Role of Enterohaemorrhagic Escherichia coli Shiga Toxin-associated Extracellular Vesicles in Host Cells.

Enterohemorrhagic Escherichia coli (EHEC) is a specific subset of Shiga toxin-producing Escherichia coli (STEC) strains that are characterized by their ability to cause bloody diarrhea (hemorrhagic colitis) and potentially life-threatening, extraintestinal complications such as hemolytic uremic syndrome (HUS), which is associated with acute renal failure., contributing to severe clinical outcomes. The Shiga toxins (Stxs), produced by EHEC, are primary virulence factors. These potent cytotoxins are composed of one enzymatically active A subunit (StxA) and five receptor-binding B subunits (StxB). Although the toxins are primarily associated with cytotoxic effects, they also elicit other pathogenic consequences due to their induction of a number of biological processes, including apoptosis through ER-stress, pro-inflammatory responses, autophagy, and post-translational modification (PTM). Moreover, several studies have reported the association between Stxs and extracellular vesicles (EVs), including microvesicles and exosomes, demonstrating that Stx-containing EVs secreted by intoxicated macrophages are taken up by recipient cells, such as toxin-sensitive renal proximal tubular epithelial cells. This mechanism likely contributes to the spreading of Stxs within the host, and may exacerbate gastrointestinal illnesses and kidney dysfunction. In this review, we summarize recent findings relating to the host responses, in different types of cells in vitro and in animal models, mediated by Stxs-containing exosomes. Due to their unique properties, EVs have been explored as therapeutic agents, drug delivery systems, and diagnostic tools. Thus, potential therapeutic applications of EVs in EHEC Stxs-mediated pathogenesis are also briefly reviewed.

Particulate matter 10 exposure affects intestinal functionality in both inflamed 2D intestinal epithelial cell and 3D intestinal organoid models.

Background: A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. Objective: The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. Methods: In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Results: Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. Conclusions: According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.

Fatal outcome of severe fever with thrombocytopenia syndrome (SFTS) and severe and critical COVID-19 is associated with the hyperproduction of IL-10 and IL-6 and the low production of TGF-ß.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause the hyperproduction of inflammatory cytokines, which have pathological effects in patient including severe or fatal cytokine storms. To characterize the effect of SFTSV and SARS-CoV-2 infection on the production of cytokines in severe fever with thrombocytopenia syndrome (SFTS) and COVID-19 patients, we performed an analysis of cytokines in SFTS and COVID-19 patients and also investigated the role of interleukin-10 (IL-10) in vitro studies: lipopolysaccharide-induced THP-1-derived macrophages, SFTSV infection of THP-1 cells, and SARS-CoV-2 infection of THP-1 cells. In this study, we found that levels of both IL-10 and IL-6 were significantly elevated, the level of transforming growth factor-ß (TGF-ß) was significantly decreased and IL-10 was elevated earlier than IL-6 in severe and critical COVID-19 and fatal SFTS patients, and inhibition of IL-10 signaling decreased the production of IL-6 and elevated that of TGF-ß. Therefore, the hyperproduction of IL-10 and IL-6 and the low production of TGF-ß have been linked to cytokine storm-induced mortality in fatal SFTS and severe and critically ill COVID-19 patients and that IL-10 can play an important role in the host immune response to severe and critical SARS-CoV-2 and fatal SFTSV infection.

Pre-existing Immunity to Endemic Human Coronaviruses Does Not Affect the Immune Response to SARS-CoV-2 Spike in a Murine Vaccination Model.

Endemic human coronaviruses (HCoVs) have been evidenced to be cross-reactive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although a correlation exists between the immunological memory to HCoVs and coronavirus disease 2019 (COVID-19) severity, there is little experimental evidence for the effects of HCoV memory on the efficacy of COVID-19 vaccines. Here, we investigated the Ag-specific immune response to COVID-19 vaccines in the presence or absence of immunological memory against HCoV spike Ags in a mouse model. Pre-existing immunity against HCoV did not affect the COVID-19 vaccine-mediated humoral response with regard to Ag-specific total IgG and neutralizing Ab levels. The specific T cell response to the COVID-19 vaccine Ag was also unaltered, regardless of pre-exposure to HCoV spike Ags. Taken together, our data suggest that COVID-19 vaccines elicit comparable immunity regardless of immunological memory to spike of endemic HCoVs in a mouse model.

Epigenetic regulation of SMAD3 by histone methyltransferase SMYD2 promotes lung cancer metastasis.

Epigenetic alterations, especially histone methylation, are key factors in cell migration and invasion in cancer metastasis. However, in lung cancer metastasis, the mechanism by which histone methylation regulates metastasis has not been fully elucidated. Here, we found that the histone methyltransferase SMYD2 is overexpressed in lung cancer and that knockdown of SMYD2 could reduce the rates of cell migration and invasion in lung cancer cell lines via direct downregulation of SMAD3 via SMYD2-mediated epigenetic regulation. Furthermore, using an in vitro epithelial-mesenchymal transition (EMT) system with a Transwell system, we generated highly invasive H1299 (In-H1299) cell lines and observed the suppression of metastatic features by SMYD2 knockdown. Finally, two types of in vivo studies revealed that the formation of metastatic tumors by shSMYD2 was significantly suppressed. Thus, we suggest that SMYD2 is a potential metastasis regulator and that the development of SMYD2-specific inhibitors may help to increase the efficacy of lung cancer treatment.

Recent Advancements in Technologies to Detect Enterohaemorrhagic Escherichia coli Shiga Toxins.

Shiga toxin (Stxs)-producing enterohaemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are major causative agents of severe bloody diarrhea (known as hemorrhagic colitis) and hemolytic uremic syndrome (HUS) associated with extraintestinal complications such as acute renal failure and neurologic impairment in infected patients under 9 years of age. Extreme nephrotoxicity of Stxs in HUS patients is associated with severe outcomes, highlighting the need to develop technologies to detect low levels of the toxin in environmental or food samples. Currently, the conventional polymerase chain reaction (PCR) or immunoassay is the most broadly used assay to detect the toxin. However, these assays are laborious, time-consuming, and costly. More recently, numerous studies have described novel, highly sensitive, and portable methods for detecting Stxs from EHEC. To contextualize newly emerging Stxs detection methods, we briefly explain the basic principles of these methods, including lateral flow assays, optical detection, and electrical detection. We subsequently describe existing and newly emerging rapid detection technologies to identify and measure Stxs.

Pseudomonas stutzeri PM101005 inhaled with atmospheric particulate matter induces lung damage through inflammatory responses.

Atmospheric particulate matter (PM) contains a mixture of chemical and biological elements that pose threat to human health by increasing susceptibility to respiratory diseases. Although the identification of the microorganisms composing the PM has been assessed, their immunological impacts are still questionable. Here, we examined the mechanisms responsible for the pathogenicity of Pseudomonas stutzeri PM101005 (PMPS), a bacterium isolated from fine dust, in lung epithelial cells, alveolar cells, and macrophages. Relative to its comparative strain Pseudomonas stutzeri (PS), infections with PMPS induced higher production of inflammatory cytokines and chemokines, mediated by the activation of NF-κB and MAPK signaling pathways. Additionally, with three-dimensional (3D) airway spheroids which mimic the human bronchial epithelium, we confirmed that PMPS infections lead to relatively higher induction of pro-inflammatory cytokines than PM infections. Consistent results were observed in murine models as the infections with PMPS provoked greater inflammatory responses than the infections with PS. These PMPS-induced responses were mediated by the signaling pathways of the Toll-like receptors (TLRs), which regulated PMPS infection and played an important role in the expression of the antibiotic peptide ß-defensin 3 (BD3) that suppressed PMPS proliferation. Moreover, PM pretreatment enhanced inflammatory responses and tissue damage of PMPS, while reducing BD3 expression. Overall, these results indicate that PM-isolated PMPS induce TLR-mediated inflammatory responses in lung tissues, and contributes to the understanding of the etiology of PM-induced respiratory damage.

Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages.

Although many cohort studies have reported that long-term exposure to particulate matter (PM) can cause lung cancer, the molecular mechanisms underlying the PM-induced increase in cancer metastasis remain unclear. To determine whether PM contributes to cancer metastasis, cancer cells were cultured with conditioned medium from PM-treated THP1 cells, and the migration ability of the treated cancer cells was assessed. The key molecules involved were identified using RNA-seq analysis. In addition, metastatic ability was analyzed in vivo by injection of cancer cells into the tail vein and intratracheal injection of PM into the lungs of C57BL/6 mice. We found that PM enhances the expression of heparin-binding EGF-like growth factor (HBEGF) in macrophages, which induces epithelial-to-mesenchymal transition (EMT) in cancer cells, thereby increasing metastasis. Macrophage stimulation by PM results in activation and subsequent nuclear translocation of the aryl hydrocarbon receptor and upregulation of HBEGF. Secreted HBEGF activates EGFR on the cancer cell surface to induce EMT, resulting in increased migration and invasion in vitro and increased metastasis in vivo. Therefore, our study reveals a critical PM-macrophage-cancer cell signaling axis mediating EMT and metastasis and provides an effective therapeutic approach for PM-induced malignancy.

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Extracellular nucleoprotein exacerbates influenza virus pathogenesis by activating Toll-like receptor 4 and the NLRP3 inflammasome.

Host immune responses, such as those initiated by pattern recognition receptor (PRR) activation, are important for viral clearance and pathogenesis. However, little is known about the interactions of viral proteins with surface PRRs or, more importantly, the association of innate immune activation with viral pathogenesis. In this study, we showed that internal influenza virus proteins were released from infected cells. Among these proteins, nucleoprotein (NP) played a critical role in viral pathogenesis by stimulating neighboring cells through toll-like receptor (TLR)2, TLR4, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. Through the activation of these PRRs, NP induced the production of interleukin (IL)-1ß and IL-6, which subsequently led to the induction of trypsin. Trypsin induced by NP increased the infectivity of influenza virus, leading to increases in viral replication and pathology upon subsequent viral infection. These results reveal the role of released NP in influenza pathogenesis and highlight the importance of the interactions of internal viral proteins with PRRs in the extracellular compartment during viral pathogenesis.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer.

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Cellular response of lung fibroblasts and epithelial cells to particulate matter10 treatment examined via comparative transcriptome analysis.

Particulate matter (PM) can be categorized by particle size (PM10, PM2.5 and PM1.0), which is an important factor affecting the biological response. Exposure to PM in the air (dust, smoke, dirt and biological contaminants) is clearly associated with lung disease (lung cancer, pneumonia and asthma). Although PM primarily affects lung epithelial cells, the specific response of related cell types to PM remains to be elucidated. The present study performed Gene Ontology (GO) analysis programs (Clustering GO and Database for Annotation, Visualization and Integrated Discovery) on differentially expressed genes in lung epithelial cells (WI­38 VA­13) and fibroblasts (WI­38) following treatment with PM10 and evaluated the cell­specific biological responses related to cell proliferation, apoptosis, adhesion and extracellular matrix production. The results suggested that short­ or long­term exposure to PM may affect cell condition and may consequently be related to several human diseases, including lung cancer and cardiopulmonary disease.

Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host.

Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC) are the major virulence factors responsible for hemorrhagic colitis, which can lead to life-threatening systemic complications including acute renal failure (hemolytic uremic syndrome) and neuropathy. Here, we report that O-GlcNAcylation, a type of post-translational modification, was acutely increased upon induction of endoplasmic reticulum (ER) stress in host cells by Stxs. Suppression of the abnormal Stx-mediated increase in O-GlcNAcylation effectively inhibited apoptotic and inflammatory responses in Stx-susceptible cells. The protective effect of O-GlcNAc inhibition for Stx-mediated pathogenic responses was also verified using three-dimensional (3D)-cultured spheroids or organoids mimicking the human kidney. Treatment with an O-GlcNAcylation inhibitor remarkably improved the major disease symptoms and survival rate for mice intraperitoneally injected with a lethal dose of Stx. In conclusion, this study elucidates O-GlcNAcylation-dependent pathogenic mechanisms of Stxs and demonstrates that inhibition of aberrant O-GlcNAcylation is a potential approach to treat Stx-mediated diseases.

Enhanced Large-Scale Production of Hahella chejuensis-Derived Prodigiosin and Evaluation of Its Bioactivity.

Prodigiosin as a high-valued compound, which is a microbial secondary metabolite, has the potential for antioxidant and anticancer effects. However, the large-scale production of functionally active Hahella chejuensis-derived prodigiosin by fermentation in a cost-effective manner has yet to be achieved. In the present study, we established carbon source-optimized medium conditions, as well as a procedure for producing prodigiosin by fermentation by culturing H. chejuensis using 10 L and 200 L bioreactors. Our results showed that prodigiosin productivity using 250 ml flasks was higher in the presence of glucose than other carbon sources, including mannose, sucrose, galactose, and fructose, and could be scaled up to 10 L and 200 L batches. Productivity in the glucose (2.5 g/l) culture while maintaining the medium at pH 6.89 during 10 days of cultivation in the 200 L bioreactor was measured and increased more than productivity in the basal culture medium in the absence of glucose. Prodigiosin production from 10 L and 200 L fermentation cultures of H. chejuensis was confirmed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses for more accurate identification. Finally, the anticancer activity of crude extracted prodigiosin against human cancerous leukemia THP-1 cells was evaluated and confirmed at various concentrations. Conclusively, we demonstrate that culture conditions for H. chejuensis using a bioreactor with various parameters and ethanol-based extraction procedures were optimized to mass-produce the marine bacterium-derived high purity prodigiosin associated with anti-cancer activity.

Escherichia coli Shiga Toxins and Gut Microbiota Interactions.

Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are enterohemorrhagic bacteria that induce hemorrhagic colitis. This, in turn, may result in potentially lethal complications, such as hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, acute renal failure, and neurological abnormalities. Both species of bacteria produce Shiga toxins (Stxs), a phage-encoded exotoxin inhibiting protein synthesis in host cells that are primarily responsible for bacterial virulence. Although most studies have focused on the pathogenic roles of Stxs as harmful substances capable of inducing cell death and as proinflammatory factors that sensitize the host target organs to damage, less is known about the interface between the commensalism of bacterial communities and the pathogenicity of the toxins. The gut contains more species of bacteria than any other organ, providing pathogenic bacteria that colonize the gut with a greater number of opportunities to encounter other bacterial species. Notably, the presence in the intestines of pathogenic EHEC producing Stxs associated with severe illness may have compounding effects on the diversity of the indigenous bacteria and bacterial communities in the gut. The present review focuses on studies describing the roles of Stxs in the complex interactions between pathogenic Shiga toxin-producing E. coli, the resident microbiome, and host tissues. The determination of these interactions may provide insights into the unresolved issues regarding these pathogens.

EHMT1 knockdown induces apoptosis and cell cycle arrest in lung cancer cells by increasing CDKN1A expression.

Dozens of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases such as cancer remain largely unclear. Here, we demonstrate the involvement of EHMT1, a histone lysine methyltransferase, in lung cancer. Immunohistochemical analysis indicated that the expression levels of EHMT1 are significantly elevated in human lung carcinomas compared with non-neoplastic lung tissues. Through gene ontology analysis of RNA-seq results, we showed that EHMT1 is clearly associated with apoptosis and the cell cycle process. Moreover, FACS analysis and cell growth assays showed that knockdown of EHMT1 induced apoptosis and G1 cell cycle arrest via upregulation of CDKN1A in A549 and H1299 cell lines. Finally, in 3D spheroid culture, compared to control cells, EHMT1 knockdown cells exhibited reduced aggregation of 3D spheroids and clear upregulation of CDKN1A and downregulation of E-cadherin. Therefore, the results of the present study suggest that EHMT1 plays a critical role in the regulation of cancer cell apoptosis and the cell cycle by modulating CDKN1A expression. Further functional analyses of EHMT1 in the context of human tumorigenesis may aid in the development of novel therapeutic strategies for cancer.

Effects of Hahella chejuensis-Derived Prodigiosin on UV-Induced ROS Production, Inflammation and Cytotoxicity in HaCaT Human Skin Keratinocytes.

Prodigiosins, which are natural tripyrrole red pigments and synthetic derivatives, reportedly have multiple biological effects mainly on various types of cancer cells. However, the effects of bacterial prodigiosin on non-cancerous HaCaT human skin keratinocytes have not been reported. Therefore, the present study aimed to investigate the functional activities of prodigiosin derived from cultures of the bacterium Hahella chejuensis in HaCaT cells. Cell viability, the cell proliferation rate, and reactive oxygen species (ROS) production in vitro were assayed following treatment of HaCaT cells with prodigiosin. Prodigiosin did not cause cytotoxicity and notably increased proliferation of HaCaT cells. Furthermore, prodigiosin reduced ultraviolet (UV) irradiation-induced ROS production and the inflammatory response in HaCaT cells. More importantly, prodigiosin reduced matrix metalloproteinase-9 expression and increased collagen synthesis in UV-irradiated HaCaT cells, demonstrating that it elicits anti-aging effects. In conclusion, our results reveal that H. chejuensis-derived prodigiosin is a potential natural product to develop functional cosmetic ingredients.

Loss of desmoglein-2 promotes gallbladder carcinoma progression and resistance to EGFR-targeted therapy through Src kinase activation.

Gallbladder carcinoma (GBC) exhibits poor prognosis due to local recurrence, metastasis, and resistance to targeted therapies. Using clinicopathological analyses of GBC patients along with molecular in vitro and tumor in vivo analysis of GBC cells, we showed that reduction of Dsg2 expression was highly associated with higher T stage, more perineural, and lymphatic invasion. Dsg2-depleted GBC cells exhibited significantly enhanced proliferation, migration, and invasiveness in vitro and tumor growth and metastasis in vivo through Src-mediated signaling activation. Interestingly, Dsg2 binding inhibited Src activation, whereas its loss activated cSrc-mediated EGFR plasma membrane clearance and cytoplasmic localization, which was associated with acquired EGFR-targeted therapy resistance and decreased overall survival. Inhibition of Src activity by dasatinib enhanced therapeutic response to anti-EGFR therapy. Dsg2 status can help stratify predicted patient response to anti-EGFR therapy and Src inhibition could be a promising strategy to improve the clinical efficacy of EGFR-targeted therapy.