Utilization of Macroalgae for the Production of Bioactive Compounds and Bioprocesses Using Microbial Biotechnology.

To achieve sustainable development, alternative resources should replace conventional resources such as fossil fuels. In marine ecosystems, many macroalgae grow faster than terrestrial plants. Macroalgae are roughly classified as green, red, or brown algae based on their photosynthetic pigments. Brown algae are considered to be a source of physiologically active substances such as polyphenols. Furthermore, some macroalgae can capture approximately 10 times more carbon dioxide from the atmosphere than terrestrial plants. Therefore, they have immense potential for use in the environment. Recently, macroalgae have emerged as a biomass feedstock for bioethanol production owing to their low lignin content and applicability to biorefinery processes. Herein, we provided an overview of the bioconversion of macroalgae into bioactive substances and biofuels using microbial biotechnology, including engineered yeast designed using molecular display technology.

Progress of Molecular Display Technology Using Saccharomyces cerevisiae to Achieve Sustainable Development Goals.

In the long history of microorganism use, yeasts have been developed as hosts for producing biologically active compounds or for conventional fermentation. Since the introduction of genetic engineering, recombinant proteins have been designed and produced using yeast or bacterial cells. Yeasts have the unique property of expressing genes derived from both prokaryotes and eukaryotes. Saccharomyces cerevisiae is one of the well-studied yeasts in genetic engineering. Recently, molecular display technology, which involves a protein-producing system on the yeast cell surface, has been established. Using this technology, designed proteins can be displayed on the cell surface, and novel abilities are endowed to the host yeast strain. This review summarizes various molecular yeast display technologies and their principles and applications. Moreover, S. cerevisiae laboratory strains generated using molecular display technology for sustainable development are described. Each application of a molecular displayed yeast cell is also associated with the corresponding Sustainable Development Goals of the United Nations.

Construction of HGF-Displaying Yeast by Cell Surface Engineering.

Hepatocyte growth factor (HGF) has been investigated as a regulator for immune reactions caused by transplantation and autoimmune diseases and other biological functions. Previous studies demonstrated that cDNA-encoding HGF administration could inhibit acute graft-versus-host disease (GVHD) after treatment via hematopoietic stem cell transplantation. This study aimed to show the preparation of HGF protein on yeast cell surfaces to develop a tool for the oral administration of HGF to a GVHD mouse model. In this study, full-length HGF and the heavy chain of HGF were genetically fused with α-agglutinin and were successfully displayed on the yeast cell surface. This study suggested that yeast cell surface display engineering could provide a novel administration route for HGF.

Functional Evaluation of Anti-TNF-α Affibody Molecules in Biochemical Detection and Inhibition to Signalling Pathways of a Synovial Cell.

BACKGROUND: An affibody molecule obtained from a bioengineered staphylococcal protein was previously shown to act as an affinity binder for a wide range of targets and develop Tumour Necrosis Factor α (TNF-α)-binding clones. METHODS: In this study, we demonstrated that affibody molecules against TNF-α could bind to recombinant TNF-α on the membrane for biochemical detection. In addition, we examined whether the affibody molecules could block binding between recombinant TNF-α and its receptor on MH7A synovial cells. RESULTS: When a TNF-α-binding affibody was added, the production level of inflammatory mediators IL-6 and MMP-3 in MH7A were found to decrease up to 44%. Additionally, proliferation of synovial cells was also inhibited by the addition of TNF-α to cultivation media. CONCLUSION: These results suggest that affibody molecules against TNF-α could be candidate molecules for the detection of TNF-α during biochemical analysis and pharmacotherapy for rheumatoid arthritis.

Molecular and Physiological Study of Candida albicans by Quantitative Proteome Analysis.

Candida albicans is one of the major pathogens that cause the serious infectious condition known as candidiasis. C. albicans was investigated by proteome analysis to systematically examine its virulence factors and to promote the development of novel pharmaceuticals against candidiasis. Here, we review quantitative time-course proteomics data related to C. albicans adaptation to fetal bovine serum, which were obtained using a nano-liquid chromatography/tandem mass spectrometry system equipped with a long monolithic silica capillary column. It was revealed that C. albicans induced proteins involved in iron acquisition, detoxification of oxidative species, energy production, and pleiotropic stress tolerance. Native interactions of C. albicans with macrophages were also investigated with the same proteome-analysis system. Simultaneous analysis of C. albicans and macrophages without isolating individual living cells revealed an attractive strategy for studying the survival of C. albicans. Although those data were obtained by performing proteome analyses, the molecular physiology of C. albicans is discussed and trials related to pharmaceutical applications are also examined.

Small RNAs detected in exosomes derived from the MH7A synovial fibroblast cell line with TNF-α stimulation.

Rheumatoid arthritis (RA) is an autoimmune disease that causes the chronic inflammation of the joints. Intercellular communication containing synovial fibroblasts seems to play a major role in RA pathogenesis. In this study, to better understand intercellular communication related to RA pathogenesis, we identified exosomal microRNAs (miRNAs) derived from synovial fibroblasts. Exosomes were collected from an RA synovial fibroblast (RASF) cell line, namely, MH7A, with or without stimulation by tumor necrosis factor alpha (TNF-α). We used small RNA sequencing to analyze the profile of small RNAs, including miRNAs, in MH7A exosomes and cells. By using differential expression analysis, we identified four miRNAs (miR-155-5p, miR-146a-5p, miR-323a-5p, and miR-1307-3p) that are upregulated in exosomes with TNF-α stimulation. The identification of miR-155-5p and miR-146a-5p which have been reported in RA patients demonstrated the validity of our experimental model. Other two miRNAs were newly identified. miR-323a-5p was predicted to target the protein encoding gene CD6, which attenuates T-cell activation signals, and miR-1307-3p was predicted to target the protein encoding gene N-myc downstream-regulated gene 2 (NDRG2), which inhibits osteoclast-related gene expression. The results suggested that these miRNAs might be involved in RA pathogenesis. We hope our results will help us understand the role of RASF exosomes in RA pathogenesis.

Preparation of an Oral Vaccine by Proteome Analysis and Molecular Display Technology.

In recent years, genetic engineering and protein expression technologies have promoted the development of recombinant protein vaccines. To accelerate the development of efficient vaccines for mycosis, screening candidate antigens, and determining the optimal route of administration are indispensable steps. Two methods for identifying novel antigens and producing antigens specific to Candida albicans, as a model causative pathogen of mycosis, are discussed in this chapter. Specifically, the application of liquid chromatography/tandem mass spectrometry using a long monolithic column for proteome analysis to identify virulence factors of C. albicans, followed by molecular display technology to produce an oral vaccine using antigens found by the proteomic study, is described.

The Impact of MicroRNA-223-3p on IL-17 Receptor D Expression in Synovial Cells.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting joints. Elevated plasma levels of microRNA-223-3p (miR-223-3p) in patients with RA are implicated in the pathogenesis of the disease. This study aimed to analyze the functional role of miR-223-3p in the pathogenesis of RA by overexpressing miR-223-3p in synovial cell lines. METHODS: Arthritis was induced in the RA model of SKG mice by injection of ß-glucan. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Plasma levels of miRNA were determined by panel real-time PCR analysis. Target genes of the differentially expressed miRNAs in SKG mice were analyzed using miRNA target prediction algorithms. The dual-luciferase reporter system was used to evaluate the relationship between miR-223-3p and IL-17 receptor D (IL-17RD). The activity of miR-223-3p was analyzed by transfection of plasmid vectors overexpressing miR-223-3p into IL-17RD-expressing NIH3T3 and MH7A cell lines. Il6 and Il17rd mRNA expression was analyzed by quantitative real-time PCR. IL-17RD protein expression was analyzed by western blot analysis. RESULTS: We identified 17 upregulated miRNAs (fold change > 2.0) in plasma of SKG mice injected with ß-glucan relative to untreated SKG mice. Il17rd was identified as the candidate target gene of miR-223-3p using five miRNA target prediction algorithms. The transfection of plasmid vectors overexpressing miR-223-3p into NIH3T3 and MH7A cells resulted in the downregulation of Il17rd expression and upregulation of Il6 expression. Expression of miR-223-3p and Il6 mRNA in MH7A cells was upregulated; however, that of Il17rd mRNA was downregulated following TNF-α stimulation. IL-17RD expression in synovial tissues from SKG mice and RA patients was inversely correlated with the severity of arthritis. CONCLUSION: This study is the first to demonstrate that miR-223-3p downregulates IL-17RD in both mouse and human cells; miR-223-3p may contribute to the pathogenesis of RA by downregulating the expression of IL-17RD and upregulating that of IL-6 in synovial cells.

A comparative proteomics study of a synovial cell line stimulated with TNF-α.

To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor-α (TNF-α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF-α. Next, we selected 269 differentially produced proteins that were detected only under TNF-α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF-α-stimulated MH7A cells, we observed substantial production of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins such as BH3-interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase-3. These results indicate that the upregulation of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins in synovial cells in response to TNF-α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.

Oral Vaccine Development by Molecular Display Methods Using Microbial Cells.

Oral vaccines are easier to administer than injectable vaccines. To induce an adequate immune response using vaccines, antigenic proteins are usually combined with adjuvant materials. This chapter presents methodologies for the design of oral vaccines using molecular display technology. In molecular display technology, antigenic proteins are displayed on a microbial cell surface with adjuvant ability. This technology would provide a quite convenient process to produce oral vaccines when the DNA sequence of an efficient antigenic protein is available. As an example, oral vaccines against candidiasis were introduced using two different molecular display systems with Saccharomyces cerevisiae and Lactobacillus casei.

Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation.

Candida albicans is an opportunistic pathogen that causes fatal diseases in immunocompromised hosts. Host resistance against C. albicans relies on ingestion of the pathogen by macrophages. Analysis of the escaping behavior of C. albicans from macrophages is required to understand the onset of systemic candidiasis. In this study, native interactions of C. albicans with macrophages were investigated by proteome analysis using high efficiency of long monolithic silica capillary column. Using this system, we developed a method of "mixed and quantitative proteome analysis" in which C. albicans and macrophages were simultaneously analyzed by nanoLC-MS/MS without the need to isolate the two individual living cells. Two hundred twenty-seven proteins from C. albicans and five proteins from macrophages were identified as candidate interaction-specific molecules. C. albicans seemed to produce glucose through a ß-oxidation pathway, a glyoxylate cycle, and gluconeogenesis for escape from macrophages. Up-regulation of stress-related and candidate pathogenic proteins in C. albicans indicated how C. albicans endured the harsh environment inside the macrophages. Down-regulation of apoptosis-associated protein NOA1- and chaperone HSPA1A-syntheses in macrophage indicated that C. albicans was able to escape from macrophages in part by suppressing the production of these macrophage proteins.

Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.

We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3K-AKT-Smad pathways. Therefore, blocking c-Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA.

Differential regulation of c-Met signaling pathways for synovial cell function.

We previously demonstrated that blocking the hepatocyte growth factor (HGF) receptor, c-Met, using a HGF antagonist, NK4, inhibited arthritis in a rheumatoid arthritis (RA) model mice. In the present study, we investigated the role of c-Met signaling in synovial cell function. We demonstrated that synovial tissues from RA patients and MH7A cells, a human RA synovial cell line, expressed HGF and c-Met. HGF and c-Met expression in RA synovium was increased compared to osteoarthritis synovium suggesting increased c-Met signaling in RA synovial cells. The c-Met inhibitor, SU11274, inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated MH7A cells. MEK and PI3K inhibitors suppressed production of matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) by MH7A cells, suggesting that c-Met-MEK-ERK and c-Met-PI3K-AKT pathways are involved positively regulating MH7A cell function. Although SU11274 suppressed MMP-3 and VEGF production it enhanced PGE2 production by MH7A cells suggesting that negative regulation by c-Met signaling, independent of the MEK-ERK and PI3K-AKT pathways, is involved in PGE2 production. Blocking c-Met signaling may be therapeutically useful to inhibit angiogenesis and cartilage and bone destruction by inhibiting VEGF and MMP-3 production, while enhancing PGE2 production in synovial cells in RA.

Evaluation of Mdh1 protein as an antigenic candidate for a vaccine against candidiasis.

Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.

Inhibitory effects of H-Ras/Raf-1-binding affibody molecules on synovial cell function.

Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.

Bioadsorption strategies with yeast molecular display technology.

Molecular display techniques using microbial cell surfaces have been widely developed in the past twenty years, and are useful tools as whole cell catalysts for various applications such as bioconversion, bioremediation, biosensing, and the screening system of protein libraries. Furthermore, different types of microbial cells among eukaryotic and prokaryotic strains have been investigated for their use in surface display technologies. Recently, several kinds of protein-displaying yeasts have been utilized as bioadsorbents in this platform technology. In particular, these trials have successfully expanded the possibility of applications to metal binding, affinity purification, and receptor-ligand interaction by using the yeast cell surface. In this mini review, we describe the general principles of molecular display technology using yeast cells and its applications, with a particular focus on bioadsorption.

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

Biochemical analysis and application of molecular display technology on Candida albicans for diagnosing and preventing candidiasis.

Medical facilities and advances in therapeutics have improved world over in recent times. Concomitant with this, the human population has been growing steadily. However, emerging infectious diseases such as severe acute respiratory syndrome (SARS) and AIDS, as well as re-emerging infectious diseases such as Japanese encephalitis and dengue fever, have been spreading in recent times. Three major infectious diseases, namely AIDS, malaria, and tuberculosis, are killing around 8 million people in the world annually. Although drugs effective against these infectious diseases are available at present, drastic therapeutics have not been developed yet. In addition, vaccines against these diseases often cannot prevent infections, because pathogenic viruses or bacteria evade the immune system of the host. Many diseases and emerging infections of pathogenic bacteria cannot be controlled by conventional pharmaceutics. These pathogens secrete regulatory factors. When the produced regulatory factor attains a certain level, an active factor is then produced by the pathogen to destroy the host. Considering these phenomena, we thought investigating characteristic regulatory or active factors will pave the way for developing novel vaccines or diagnostic drugs. Therefore, candidiasis was selected as a model, and application of the secretory protease of Candida albicans was examined for the development of novel drugs. Screening of novel candidates of antigens of C. albicans and vaccine development are also underway. In this paper, our strategy of platform technology against various infectious diseases are introduced.

Hepatocyte growth factor regulates immune reactions caused by transplantation and autoimmune diseases.

Hepatocyte growth factor (HGF) was first identified and cloned as a mitogenic protein for hepatocytes, and subsequent studies revealed that HGF has multiple biological effects on a wide variety of cells, including mitogenic, motogenic, morphogenic, anti-apoptotic, and angiogenic activities. It plays roles in organizing tissues during development and regeneration. HGF may be applied for the treatment of acute onset diseases such as fulminant hepatitis, myocardial infarction, acute renal failure, cerebral infarction, and chronic diseases like liver cirrhosis, chronic renal failure, pulmonary fibrosis, cardiomyopathy, and arteriosclerosis obliterans. HGF also has immunomodulatory activities and we previously demonstrated that its administration inhibited acute graft-versus-host disease (GVHD) after treatment with hematopoietic stem cell transplantation. We also demonstrated that HGF inhibited lupus nephritis induced by chronic GVHD and dermal sclerosis in systemic sclerosis using model mice. More than 7 hundred thousand patients suffer from rheumatoid arthritis (RA) in Japan. Although the prognosis of these patients has improved by the treatment of biological agents such as TNF-α and IL-6 blockers, there remain many for whom these agents have not proved beneficial. Recently, using RA model mice, we demonstrated that the HGF antagonist, NK4, can block disease progression of RA through its anti-angiogenic and immunomodulatory actions. In this review article, we discuss the possible roles of HGF signaling for the treatment of immunological reactions in transplantation and autoimmune diseases.