Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae

The vacuoles of the yeast Saccharomyces cerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa B signaling pathway.     

Evaluation of a fosmid-clone-based microarray for comparative analysis of swine fecal metagenomes

Glass slide arrayed with fosmid clone DNAs generated from swine feces as probes were fabricated and used as a metagenome microarray (MGA). MGA appeared to be specific to their corresponding target genomic fragments. The detection limit was 10 ng of genomic DNA (ca. 10(6) bacterial cells) in the presence of 1000 ng of background DNA. Linear relationships between the signal intensity and the target DNA (20-100 ng) were observed (r ( 2 )=0.98). Application of MGA to the comparison of swine fecal metagenomes suggested that the microbial community composition of swine intestine could be dependent on the health state of swine.     

Comparative genomic analysis of <Emphasis Type="Italic">Geosporobacter ferrireducens</Emphasis> and its versatility of anaerobic energy metabolism

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H⁺ and Na⁺ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na⁺-dependent F-type ATPase and H⁺-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.     

Effects of different mineral supplements on fertilization of phenol-contaminated soils by Corynebacterium glutamicum

This study focused on the effects of different mineral supplements on the ability of Corynebacterium glutamicum to degrade phenol in contaminated soil and convert the phenol into useful amino acids. Three types of minerals including FeSO₄, MgSO₄, and MnSO₄ were added at several concentrations to C. glutamicum culture media containing 1% yeast extract prior to treating the soil samples with 4.24 mM phenol. The reactor was incubated at 30°C and 150 rpm for 3 days, and the treated soil was sampled daily and analyzed using gas chromatography for residual phenol and the amino acids produced. Additionally, a plant toxicity assay was employed to examine the fertilization of the phenol-contaminated soil after C. glutamicum treatment supplemented with the three minerals. Our results suggested that among various tested concentrations, 72 μM of iron showed a significant effect on the utilization of phenol by C. glutamicum for conversion to amino acids, therefore enhancing fertilization of the phenol-contaminated soil.     

Enhanced immune response by vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 macrophages

Vacuoles are membrane vesicles in eukaryotic cells, the digestive system of cells that break down substances absorbed outside the cell and digest the useless components of the cell itself. Researches on anticancer and intractable diseases using vacuoles are being actively conducted. The practical application of the present study to animals requires the determination of the biocompatibility of vacuole. In the present study, we evaluated the effects of vacuoles isolated from Saccharomyces cerevisiae in RAW 264.7 cells. This showed a significant increase in the production of nitric oxide (NO) produced by macrophage activity. Using Reactive Oxygen Species (ROS) assay, we identified that ROS is increased in a manner dependent on vacuole concentration. Western blot analysis showed that vacuole concentration-dependently increased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2). Therefore, iNOS expression was stimulated to induce NO production. In addition, pro-inflammatory cytokines levels promoted, such as interleukin (IL) 6 (IL-6) and tumor necrosis factor (TNF) α (TNF-α). In summary, vacuoles activate the immune response of macrophages by promoting the production of immune-mediated transporters NO, ROS, and pro-inflammatory cytokines.     

Specific histamine regulating activity of surface-modified yeast vacuoles by histamine- binding protein and its immune-enhancing effect

We aimed to develop a biocompatible material that could enhance weakened immunity and control histamine in vivo. Histamine-binding protein (HBP) vacuoles have a mechanism of action that directly binds to the histamine molecule. It is designed to eliminate the side effects of antihistamine caused by binding to other receptors. HBP vacuoles were designed to produce a material that was biocompatible, and could enhance immunity. First, a recombinant vector was designed so that HBP was located on the vacuole surface, and expressed towards the cytoplasm. The vector was transformed into yeast, and protein expression was induced. Then, the vacuole was isolated by centrifugation to complete HBP vacuoles. Cytotoxicity test was conducted for application to RAW 264.7 cells. In addition, immune enhancement reaction and histamine inhibition were confirmed through phagocytosis assay and histamine ELISA. RAW 264.7 cells were pre-treated with HBP vacuoles to confirm the immune enhancement of HBP vacuoles. As a result, it was confirmed that the immunostimulatory effect of the vacuole was increased in a concentration-dependent manner. In addition, the reduction of histamine was confirmed by treating the HBP vacuoles. As a result, HBP vacuoles reduced the histamine secreted from RAW 264.7 cells by about 75%.     

Pseudozyma jejuensis sp. nov., a novel cutinolytic ustilaginomycetous yeast species that is able to degrade plastic waste

An ustilaginomycetous anamorphic yeast, isolated from orange leaves on Jeju island in South Korea, represents a novel Pseudozyma species according to morphologic and physiologic findings and molecular taxonomic analysis using the D1/D2 domains of the large subunit (26S) rRNA gene and the internally transcribed spacer (ITS) 1+2 regions. The name Pseudozyma jejuensis sp. nov. is proposed for this novel species, with OL71(T) (=KCTC 17482(T)=CBS 10454(T)) as type strain. In the present study, we have also demonstrated that Pseudozyma jejuensis OL71 is capable of producing cutinase and degrading polycaprolactone. These results suggest that Pseudozyma jejuensis or its cutinase may be useful for the biological degradation of plastic waste.