Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

BACKGROUND: Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. RESULTS: At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10 mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. CONCLUSION: Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

Bigger problems from smaller colonies: emergence of antibiotic-tolerant small colony variants of Mycobacterium avium complex in MAC-pulmonary disease patients.

BACKGROUND: Mycobacterium avium complex (MAC) is a group of slow-growing mycobacteria that includes Mycobacterium avium and Mycobacterium intracellulare. MAC pulmonary disease (MAC-PD) poses a threat to immunocompromised individuals and those with structural pulmonary diseases worldwide. The standard treatment regimen for MAC-PD includes a macrolide in combination with rifampicin and ethambutol. However, the treatment failure and disease recurrence rates after successful treatment remain high. RESULTS: In the present study, we investigated the unique characteristics of small colony variants (SCVs) isolated from patients with MAC-PD. Furthermore, revertant (RVT) phenotype, emerged from the SCVs after prolonged incubation on 7H10 agar. We observed that SCVs exhibited slower growth rates than wild-type (WT) strains but had higher minimum inhibitory concentrations (MICs) against multiple antibiotics. However, some antibiotics showed low MICs for the WT, SCVs, and RVT phenotypes. Additionally, the genotypes were identical among SCVs, WT, and RVT. Based on the MIC data, we conducted time-kill kinetic experiments using various antibiotic combinations. The response to antibiotics varied among the phenotypes, with RVT being the most susceptible, WT showing intermediate susceptibility, and SCVs displaying the lowest susceptibility. CONCLUSIONS: In conclusion, the emergence of the SCVs phenotype represents a survival strategy adopted by MAC to adapt to hostile environments and persist during infection within the host. Additionally, combining the current drugs in the treatment regimen with additional drugs that promote the conversion of SCVs to RVT may offer a promising strategy to improve the clinical outcomes of patients with refractory MAC-PD.

Anti-heat shock protein 10 IgG in chronic spontaneous urticaria: Relation with miRNA-101-5p and platelet-activating factor.

BACKGROUND: Anti-heat shock protein (HSP) autoantibodies are detected in autoimmune diseases. We sought to ascertain whether anti-HSP10 IgG is present in patients with CSU and to elucidate the role of HSP10 in CSU pathogenesis. METHOD: Using a human proteome microarray, six potential autoantibodies had higher expression in 10 CSU samples compared with 10 normal controls (NCs). Among them, HSP10 IgG autoantibody was quantified by immune dot-blot assay in sera from 86 CSU patients and 44 NCs. The serum levels of HSP10 and microRNA-101-5p were measured in CSU patients and NCs. The effects of HSP10 and miR-101-5p on mast cell degranulation in response to IgE, compound 48/80, and platelet-activating factor (PAF) were investigated. RESULTS: CSU patients had higher IgG positivity to HSP10 (40.7% vs. 11.4%, p = .001), lower serum HSP10 levels (5.8 ± 3.6 vs. 12.2 ± 6.6 pg/mL, p < .001) than in NCs, and their urticaria severity was associated with anti-HSP10 IgG positivity, while HSP10 levels were related to urticaria control status. MiR-101-5p was increased in CSU patients. PAF enhanced IL4 production in PBMCs from CSU patients. IL-4 upregulated miR-101-5p and reduced HSP10 expression in keratinocytes. Transfection of miR-101-5p reduced HSP10 expression in keratinocytes. MiR-101-5p promoted PAF-induced mast cell degranulation, while HSP10 specifically prevented it. CONCLUSION: A new autoantibody, anti-HSP10 IgG was detected in CSU patients, which showed a significant correlation with UAS7 scores. A decreased serum HSP10 level was associated with upregulation of miR-101-5p due to increased IL-4 and PAF in CSU patients. Modulation of miR-101-5p and HSP10 may be a novel therapeutic approach for CSU.

Comparative genetic characterization of CMY-2-type beta-lactamase producing pathogenic Escherichia coli isolated from humans and pigs suffering from diarrhea in Korea.

BACKGROUND: Pathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea. RESULTS: All 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for ß-lactam/ß-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate). CONCLUSION: Our findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis.

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Delineating transcriptional crosstalk between Mycobacterium avium subsp. paratuberculosis and human THP-1 cells at the early stage of infection via dual RNA-seq analysis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic debilitating disease in ruminants. To control this disease, it is crucial to understand immune evasion and the mechanism of persistence by analyzing the early phase interplays of the intracellular pathogens and their hosts. In the present study, host-pathogen interactions at the transcriptomic level were investigated in an in vitro macrophage infection model. When differentiated human THP-1 cells were infected with MAP, the expression of various genes associated with stress responses and metabolism was altered in both host and MAP at 3 h post-infection. MAP upregulates stress-responsive global gene regulators, such as two-component systems and sigma factors, in response to oxidative and cell wall stress. Downstream genes involved in type VII secretion systems, cell wall synthesis (polyketide biosynthesis proteins), and iron uptake were changed in response to the intracellular environment of macrophages. On the host side, upregulation of inflammatory cytokine genes was observed along with pattern recognition receptor genes. Notably, alterations in gene sets involved in arginine metabolism were observed in both the host and MAP, along with significant downregulation of NOS2 expression. These observations suggest that the utilization of metabolites such as arginine by intracellular MAP might affect host NO production. Our dual RNA-seq data can provide novel insights by capturing the global transcriptome with higher resolution, especially in MAP, thus enabling a more systematic understanding of host-pathogen interactions.

Flavanols and Flavanes from Crinum asiaticum and Their Effects on LPS Signaling Pathway Through the Inhibition of NF-κB Activation.

Three new flavanols, (2R,3S)-7-methoxy-flavan-3-ol (1: ), (2R,3S)-7-hydroxy-flavan-3-ol (2: ), and (2R,3S)-2'-hydroxy-7-methoxy-flavan-3-ol (3: ), together with two known flavans (4: and 5: ), were isolated from the chloroform extract of Crinum asiaticum. Their structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-MS, and CD data. The isolated compounds 1: and 3: -5: showed inhibitory activity toward LPS-induced nitric oxide (NO) production. Further investigation of the NF-κB pathway mechanisms indicated that 1: and 3: -5: inhibited the LPS-induced IL-6 production and p65 subunit phosphorylation of NF-κB in RAW264.7 cells, with an effective dose of 10 µM.

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells to Model Myogenesis and Muscle Regeneration.

In vitro organoids derived from human pluripotent stem cells (hPSCs) have been developed as essential tools to study the underlying mechanisms of human development and diseases owing to their structural and physiological similarity to corresponding organs. Despite recent advances, there are a few methodologies for three-dimensional (3D) skeletal muscle differentiation, which focus on the terminal differentiation into myofibers and investigate the potential of modeling neuromuscular disorders and muscular dystrophies. However, these methodologies cannot recapitulate the developmental processes and lack regenerative capacity. In this study, we developed a new method to differentiate hPSCs into a 3D human skeletal muscle organoid (hSkMO). This organoid model could recapitulate the myogenesis process and possesses regenerative capacities of sustainable satellite cells (SCs), which are adult muscle stem/progenitor cells capable of self-renewal and myogenic differentiation. Our 3D model demonstrated myogenesis through the sequential occurrence of multiple myogenic cell types from SCs to myocytes. Notably, we detected quiescent, non-dividing SCs throughout the hSkMO differentiation in long-term culture. They were activated and differentiated to reconstitute muscle tissue upon damage. Thus, hSkMOs can recapitulate human skeletal muscle development and regeneration and may provide a new model for studying human skeletal muscles and related diseases.

Quercetin and Isorhamnetin Reduce Benzo[a]pyrene-Induced Genotoxicity by Inducing RAD51 Expression through Downregulation of miR-34a.

Benzo[a]pyrene (B[a]P) is metabolized in the liver into highly reactive mutagenic and genotoxic metabolites, which induce carcinogenesis. The mutagenic factors, including B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) and reactive oxygen species, generated during B[a]P metabolism can cause DNA damage, such as BPDE-DNA adducts, 8-oxo-dG, and double-strand breaks (DSBs). In this study, we mechanistically investigated the effects of quercetin and its major metabolite isorhamnetin on the repair of B[a]P-induced DNA DSBs. Whole-transcriptome analysis showed that quercetin and isorhamnetin each modulate the expression levels of genes involved in DNA repair, especially those in homologous recombination. RAD51 was identified as a key gene whose expression level was decreased in B[a]P-treated cells and increased by quercetin or isorhamnetin treatment. Furthermore, the number of γH2AX foci induced by B[a]P was significantly decreased by quercetin or isorhamnetin, whereas RAD51 mRNA and protein levels were increased. Additionally, among the five microRNAs (miRs) known to downregulate RAD51, miR-34a level was significantly downregulated by quercetin or isorhamnetin. The protective effect of quercetin or isorhamnetin was lower in cells transfected with a miR-34a mimic than in non-transfected cells, and the B[a]P-induced DNA DSBs remained unrepaired. Our results show that quercetin and isorhamnetin each upregulates RAD51 by downregulating miR-34a and thereby suppresses B[a]P-induced DNA damage.

Endoplasmic Reticulum Stress and Impairment of Ribosome Biogenesis Mediate the Apoptosis Induced by Ocimum x africanum Essential Oil in a Human Gastric Cancer Cell Line.

Background and Objectives: Gastric cancer remains a major unmet clinical problem worldwide. Although conventional medical treatments are available, their curative effects are generally unsatisfactory. Consequently, it remains necessary to search natural products for potential alternatives in treating gastric cancer patients. Ocimum x africanum Lour. is a culinary herb that has been used in folk medicine for various diseases, but little is known regarding its anti-cancer activity against gastric cancer cells. In the current study, we focus on the anti-cancer mechanisms of O. x africanum essential oil (OAEO) in the AGS human gastric cancer cell line. Materials and Methods: After OAEO treatment, AGS cell viability was evaluated by MTT assay. Cell migration and apoptotic nuclear morphology were determined by wound-healing assay and DAPI staining, respectively. Gene expression levels of apoptosis-related genes were quantified by qRT-PCR. Differential protein expression was determined with an LC-MS/MS-based proteomics approach to identify the key proteins that may be important in the anti-cancer mechanisms of OAEO on AGS cells. The chemical constituents of OAEO were identified by GC-MS analysis. Results: We found OAEO to exhibit a potent growth-inhibiting effect on AGS cells, with an IC50 value of 42.73 µg/mL. After OAEO treatment for 24 h, AGS cell migration was significantly decreased relative to the untreated control. OAEO-treated AGS cells exhibited common features of apoptotic cell death, including cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Apoptotic cell death was confirmed by qRT-PCR for apoptosis-related genes, revealing that OAEO decreased the expression of anti-apoptotic genes (BCL2 and BCL-xL) and activated pro-apoptotic genes and apoptotic caspase genes (TP53, BAX, CASP9, CASP12, and CASP3). Moreover, expression of CASP8 was not changed after treatment. Proteomic analysis revealed that OAEO may produce a signature effect on protein clusters relating to unfolded protein accumulation, thereby inducing severe ER stress and also impairing ribosome synthesis. STRING analysis revealed seven up-regulated and 11 down-regulated proteins, which were significantly associated with protein folding and ribosome biogenesis, respectively. Using GC-MS analysis, 6-methyl-5-hepten-2-one, citral, neral, and linalool were found to be the major chemical constituents in OAEO. Conclusions: Taken together, these results indicate that OAEO has a potential anti-proliferative effect on AGS cells. Our molecular findings show evidence supporting an important role of ER stress and ribosome biogenesis impairment in mediating the induction of cell death by OAEO through the mitochondrial-apoptotic pathway. This study, therefore, provides fundamental knowledge for future applications using OAEO as an alternative therapy in gastric cancer management.

Genomic diversity of Mycobacterium avium subsp. paratuberculosis: pangenomic approach for highlighting unique genomic features with newly constructed complete genomes.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic granulomatous enteropathy in ruminants. Determining the genetic diversity of MAP is necessary to understand the epidemiology and biology of MAP, as well as establishing disease control strategies. In the present study, whole genome-based alignment and comparative analysis were performed using 40 publicly available MAP genomes, including newly sequenced Korean isolates. First, whole genome-based alignment was employed to identify new genomic structures in MAP genomes. Second, the genomic diversity of the MAP population was described by pangenome analysis. A phylogenetic tree based on the core genome and pangenome showed that the MAP was differentiated into two major types (C- and S-type), which was in keeping with the findings of previous studies. However, B-type strains were discriminated from C-type strains. Finally, functional analysis of the pangenome was performed using three virulence factor databases (i.e., PATRIC, VFDB, and Victors) to predict the phenotypic diversity of MAP in terms of pathogenicity. Based on the results of the pangenome analysis, we developed a real-time PCR technique to distinguish among S-, B- and C-type strains. In conclusion, the results of our study suggest that the phenotypic differences between MAP strains can be explained by their genetic polymorphisms. These results may help to elucidate the diversity of MAP, extending from genomic features to phenotypic traits.

Identification of Ophiocordyceps gracilioides by Its Anti-tumor Effects through Targeting the NFκB-STAT3-IL-6 Inflammatory Pathway.

Although more than 400 species of Cordyceps s.l. have been identified, most have not been well explored regarding their potential for medicinal use. In this study, the profiles of constituents of ten different species of Ophiocordyceps, which is an unexplored species of Cordyceps, were analyzed and their anti-tumor effects were further examined. Although all Ophiocordyceps samples exhibited similar peak patterns, Ophiocordyceps gracilioides (O. grac) had a distinct constituent profile from the other samples. Furthermore, O. grac was the most active in suppressing the transcriptional activities of both nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription (STAT)3, and the production of interleukin (IL)-6 from breast cancer cells. This study demonstrated that O. grac is a relatively unexplored Cordyceps s.l. that may have medicinal potential to inhibit the NFκB-STAT3-IL-6 inflammatory pathway in cancer.

Heterogeneity of Helicobacter pylori bab genotypes and their association with clinical outcomes in Korean gastroduodenal patients.

In this study, we aimed to investigate the prevalence of bab genes (babA, babB, babC) at their three loci (loci A, B, and C) in Helicobacter pylori strains from varied clinical manifestations of Korean gastroduodenal patients. The overall prevalence of H. pylori Korean strains positive for babA and babB was 91.1% and 92.2%, respectively, but all strains were negative for bab C. H. pylori strains with two loci occupied (loci A and B) were the most prevalent in Korean patients (85.6%), compared to one locus occupied (14.4%) (locus A or B). Twelve bab genotypes were detected, additionally, the distribution of three bab genotypes was significantly associated with different clinical outcomes among Korean patients. The genotypes babA/babB/- and babA/babA+babB/- were significantly associated with peptic ulcer disease (PUD) (63.3%) and gastritis (GT) (33.3%) patients, respectively. In addition, we found that the babA+babB/babA+babB/- genotype was significantly associated with gastric cancer (GC) (36.7%) as compared to GT (6.7%) or PUD (6.7%) (p<0.05) patients. This study provided evidence that the bab genotypes in H. pylori Korean strains were highly variable. Interestingly, three patterns of bab genotypes were significantly different among patients with different clinical outcomes in the population at high-risk for GC.

Genetic Involvement of Mycobacterium avium Complex in the Regulation and Manipulation of Innate Immune Functions of Host Cells.

Mycobacterium avium complex (MAC), a collection of mycobacterial species representing nontuberculous mycobacteria, are characterized as ubiquitous and opportunistic pathogens. The incidence and prevalence of infectious diseases caused by MAC have been emerging globally due to complications in the treatment of MAC-pulmonary disease (PD) in humans and the lack of understating individual differences in genetic traits and pathogenesis of MAC species or subspecies. Despite genetically close one to another, mycobacteria species belonging to the MAC cause diseases to different host range along with a distinct spectrum of disease. In addition, unlike Mycobacterium tuberculosis, the underlying mechanisms for the pathogenesis of MAC infection from environmental sources of infection to their survival strategies within host cells have not been fully elucidated. In this review, we highlight unique genetic and genotypic differences in MAC species and the virulence factors conferring the ability to MAC for the tactics evading innate immune attacks of host cells based on the recent advances in genetic analysis by exemplifying M. avium subsp. hominissuis, a major representative pathogen causing MAC-PD in humans. Further understanding of the genetic link between host and MAC may contribute to enhance host anti-MAC immunity, but also provide novel therapeutic approaches targeting the pangenesis-associated genes of MAC.

An In Vitro Anti-Cancer Activity of Ocimum tenuiflorum Essential Oil by Inducing Apoptosis in Human Gastric Cancer Cell Line.

Background and Objectives: The effects of Ocimum tenuiflorum essential oil (OTEO) against gastric cancer remain unknown and merit investigation. Materials and Methods: In the present study, the anti-cancer activity of OTEO was examined in a human gastric cancer cell line (AGS). After OTEO treatment, AGS cell viability was determined by an MTT assay, and inhibition of metastasis was determined by cell migration and invasion assays. The expression of apoptosis-related genes in treated AGS cells was determined by qRT-PCR. Results: OTEO significantly decreased AGS cell viability in a dose-dependent manner (IC50 163.42 µg/mL) and effectively inhibited cell migration and invasion. Morphological examination demonstrated that OTEO induced cell shrinkage, chromatin condensation, and fragmentation, which are considered typical morphologies of apoptotic cell death. Pro-apoptotic genes (TP53, BAX, and BAK) were significantly up-regulated, while anti-apoptotic genes (BCL-2 and BCL-xL) were significantly down-regulated after treatment with OTEO. In addition, significantly increased gene expression was detected for CASP8, CASP9, and CASP3 in AGS cells exposed to OTEO. GC-MS analysis demonstrated that the major compound of OTEO was caryophyllene (25.85%) and α-pinene (11.66%). Conclusions: This in vitro study demonstrates for the first time that OTEO has potential anti-gastric cancer activity and may induce apoptosis in AGS cells through extrinsic and intrinsic pathways.

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis.

BACKGROUND: Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. METHODS: In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. RESULTS: Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. CONCLUSIONS: The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Apoptotic effect of enterodiol, the final metabolite of edible lignans, in colorectal cancer cells.

BACKGROUND: Enterodiol (END) is transformed by human intestinal bacteria from lignans contained in various whole-grain cereals, nuts, legumes, flaxseed, and vegetables. It is known to have several physiological effects, but its effects on mitogen-activated protein kinase (MAPK) signaling and apoptosis in colorectal cancer (CRC) cells have not yet been elucidated. We therefore investigated the effects of END on apoptosis in CRC cells and whether these effects are mediated via MAPK signaling. RESULTS: Cell proliferation was decreased by END treatment in a time-dependent manner. In particular, END treatment resulted in an apoptosis rate of up to 40% in CT26 cells but showed no cytotoxicity toward RAW264.7 macrophages. Treatment with END also suppressed the migration of CRC cells in a concentration-dependent manner. The phosphorylation of extracellular signal-regulated kinase (ERK), jun N-terminal kinase (JNK), and p38 was down-regulated with END treatment. Furthermore, END decreased the expression levels of anti-apoptotic proteins in CRC cells. CONCLUSION: Enterodiol inhibited the growth of CRC cells by controlling the MAPK signaling pathway involved in proliferation and apoptosis. These results demonstrate that END has an apoptotic effect in CRC cells. © 2018 Society of Chemical Industry.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

A novel Th1-type T-cell immunity-biasing effect of malate dehydrogenase derived from Mycobacterium avium subspecies paratuberculosis via the activation of dendritic cells.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative pathogen of Johne's disease in ruminants, characterized by chronic granulomatous enteritis; it also has zoonotic potential and is associated with Crohn's disease in humans. A better understanding of the mycobacterial antigens and their roles in the host immune response may facilitate the rational design of control strategies, including the development of effective vaccines and diagnostic tools. However, the functional roles of a large proportion of MAP antigens involved in modulating the host immune response remain unknown. In this study, an immunological role of MAP malate dehydrogenase (MDH, MAP2541c), an antigen that is upregulated in stress culture conditions, such as nutrient starvation and hypoxia, in polarizing naïve CD4+/CD8+ T cells toward Th1-biased T-cell immunity via the activation of dendritic cells (DCs) was identified. DCs treated with MAP MDH displayed characteristics of the activated and mature immune status, with augmented expression of cell surface molecules and pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, and IL-12p70, but not IL-10, along with a dose-dependent decrease in the antigen uptake capacity. A mechanistic investigation revealed that the observed DC maturation is mediated by the activation of JNK, ERK, and p38 MAP kinases, and the NF-κB signaling pathway. Notably, DCs activated by MAP MDH treatment promoted naïve CD4+/CD8+ T cell proliferation; in particular, they effectively polarized naïve CD4+ T cells to secrete IFN-γ and IL-2 and activate T-bet, but, unlike the LPS control, did not influence IL-5 and GATA-3. These results indicated that MAP MDH has the potential to induce the Th1 cell response via DC activation. Collectively, our data demonstrated that MAP MDH is a novel immunostimulatory antigen that drives Th1-biased T cell polarization via interactions with DCs, suggesting that MDP MDH has the potential to be an effective MAP vaccine antigen target and diagnostic marker.