Publications by authors named "Ryota Yamasaki"

19 Publications

  • Page 1 of 1

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Environ Microbiol 2021 Nov 19;23(11):7245-7254. Epub 2021 Oct 19.

Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, 16802-4400, USA.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.
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http://dx.doi.org/10.1111/1462-2920.15816DOI Listing
November 2021

Magnesium Hydroxide Nanoparticles Kill Exponentially Growing and Persister Cells by Causing Physical Damage.

Nanomaterials (Basel) 2021 Jun 16;11(6). Epub 2021 Jun 16.

Department of Health Promotion, Division of Infections and Molecular Biology, Kyushu Dental University, Kitakyushu 803-8580, Fukuoka, Japan.

Magnesium hydroxide nanoparticles are widely used in medicinal and hygiene products because of their low toxicity, environment-friendliness, and low cost. Here, we studied the effects of three different sizes of magnesium hydroxide nanoparticles on antibacterial activity: NM80, NM300, and NM700. NM80 (D = 75.2 nm) showed a higher bactericidal effect against than larger nanoparticles (D = 328 nm (NM300) or 726 nm (NM700)). Moreover, NM80 showed a high bactericidal effect against not only exponential cells but also persister cells, which are difficult to eliminate owing to their high tolerance to antibiotics. NM80 eliminated strains in which magnesium-transport genes were knocked out and exhibited a bactericidal effect similar to that observed in the wild-type strain. The bactericidal action involved physical cell damage, as confirmed using scanning electron microscopy, which showed that cells treated with NM80 were directly injured.
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http://dx.doi.org/10.3390/nano11061584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234494PMC
June 2021

Biological Effects of β-Glucans on Osteoclastogenesis.

Molecules 2021 Apr 1;26(7). Epub 2021 Apr 1.

Department of Health Promotion, Division of Infections and Molecular Biology, Kyushu Dental University, Fukuoka 803-8580, Japan.

Although the anti-tumor and anti-infective properties of β-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of β-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize β-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker's yeast, as well as β-1,3-glucan from inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of β-glucan derived from and suppressed bone resorption in vivo. However, zymosan derived from stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of β-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.
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http://dx.doi.org/10.3390/molecules26071982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036280PMC
April 2021

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

J Cell Physiol 2021 07 11;236(7):5098-5107. Epub 2020 Dec 11.

Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Fukuoka, Japan.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of β-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of β-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases.
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http://dx.doi.org/10.1002/jcp.30217DOI Listing
July 2021

Schizophyllum commune β-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

Carbohydr Polym 2021 Feb 23;253:117285. Epub 2020 Oct 23.

Division of Infections and Molecular Biology, Kyushu Dental University, 2-6-1 Manazuru, Kokurakitaku, Kitakyushu, Fukuoka 803-8580, Japan. Electronic address:

β-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of β-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.
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http://dx.doi.org/10.1016/j.carbpol.2020.117285DOI Listing
February 2021

Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm.

BMC Microbiol 2020 11 23;20(1):358. Epub 2020 Nov 23.

Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Fukuoka, 803-8580, Japan.

Background: Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis.

Results: Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation.

Conclusions: Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.
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http://dx.doi.org/10.1186/s12866-020-02034-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684882PMC
November 2020

Reactive Oxygen Species Penetrate Persister Cell Membranes of for Effective Cell Killing.

Front Cell Infect Microbiol 2020 18;10:496. Epub 2020 Sep 18.

Division of Infections and Molecular Biology, Department of Health Promotion, Kyushu Dental University, Kitakyushu, Japan.

Persister cells are difficult to eliminate because they are tolerant to antibiotic stress. In the present study, using artificially induced persister cells, we found that reactive oxygen species (ROS) have greater effects on persister cells than on exponential cells. Thus, we examined which types of ROS could effectively eliminate persister cells and determined the mechanisms underlying the effects of these ROS. Ultraviolet (UV) light irradiation can kill persister cells, and bacterial viability is markedly increased under UV shielding. UV induces the production of ROS, which kill bacteria by moving toward the shielded area. Electron spin resonance-based analysis confirmed that hydroxyl radicals are produced by UV irradiation, although singlet oxygen is not produced. These results clearly revealed that ROS sterilizes persister cells more effectively compared to the sterilization of exponential cells ( < 0.01). These ROS do not injure the bacterial cell wall but rather invade the cell, followed by cell killing. Additionally, the sterilization effect on persister cells was increased by exposure to oxygen plasma during UV irradiation. However, vapor conditions decreased persister cell sterilization by reducing the levels of hydroxyl radicals. We also verified the effect of ROS against bacteria in biofilms that are more resistant than planktonic cells. Although UV alone could not completely sterilize the biofilm bacteria, UV with ROS achieved complete sterilization. Our results demonstrate that persister cells strongly resist the effects of antibiotics and starvation stress but are less able to withstand exposure to ROS. It was shown that ROS does not affect the cell membrane but penetrates it and acts internally to kill persister cells. In particular, it was clarified that the hydroxy radical is an effective sterilizer to kill persister cells.
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http://dx.doi.org/10.3389/fcimb.2020.00496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530241PMC
May 2021

Persister Cells Resuscitate Using Membrane Sensors that Activate Chemotaxis, Lower cAMP Levels, and Revive Ribosomes.

iScience 2020 Jan 21;23(1):100792. Epub 2019 Dec 21.

Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802-4400, USA; The Huck Institute of the Life Sciences, Pennsylvania State University, University Park, PA 16802-4400, USA. Electronic address:

Persistence, the stress-tolerant state, is arguably the most vital phenotype since nearly all cells experience nutrient stress, which causes a sub-population to become dormant. However, how persister cells wake to reconstitute infections is not understood well. Here, using single-cell observations, we determined that Escherichia coli persister cells resuscitate primarily when presented with specific carbon sources, rather than spontaneously. In addition, we found that the mechanism of persister cell waking is through sensing nutrients by chemotaxis and phosphotransferase membrane proteins. Furthermore, nutrient transport reduces the level of secondary messenger cAMP through enzyme IIA; this reduction in cAMP levels leads to ribosome resuscitation and rescue. Resuscitating cells also immediately commence chemotaxis toward nutrients, although flagellar motion is not required for waking. Hence, persister cells wake by perceiving nutrients via membrane receptors that relay the signal to ribosomes via the secondary messenger cAMP, and persisters wake and utilize chemotaxis to acquire nutrients.
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http://dx.doi.org/10.1016/j.isci.2019.100792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957856PMC
January 2020

Identification of a potent indigoid persister antimicrobial by screening dormant cells.

Biotechnol Bioeng 2019 09 25;116(9):2263-2274. Epub 2019 Jun 25.

Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania.

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.
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http://dx.doi.org/10.1002/bit.27078DOI Listing
September 2019

σ -Dependent regulator DVU2956 switches Desulfovibrio vulgaris from biofilm formation to planktonic growth and regulates hydrogen sulfide production.

Environ Microbiol 2019 10 23;21(10):3564-3576. Epub 2019 May 23.

Department of Chemical Engineering, Pennsylvania State University, University Park, State College, PA, 16802, USA.

Microbiologically influenced corrosion causes $100 billion in damage per year, and biofilms formed by sulfate-reducing bacteria (SRB) are the major culprit. However, little is known about the regulation of SRB biofilm formation. Using Desulfovibrio vulgaris as a model SRB organism, we compared the transcriptomes of biofilm and planktonic cells and identified that the gene for σ -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (P ), we found production of DVU2956 inhibits biofilm formation by 70%. Corroborating this result, deleting dvu2956 increased biofilm formation, and this biofilm phenotype could be complemented. By producing proteins in biofilms from genes controlled by DVU2956 (dvu2960 and dvu2962), biofilm formation was inhibited almost completely. A second round of RNA-seq for the production of DVU2956 revealed DVU2956 influences electron transport via an Hmc complex (high-molecular-weight cytochrome c encoded by dvu0531-dvu0536) and the Fe-only hydrogenase (encoded by dvu1769, hydA and dvu1770, hydB) to control H S production. Corroborating these results, producing DVU2956 in biofilms decreased H S production by half, deleting dvu2956 increased H S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H S formation.
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http://dx.doi.org/10.1111/1462-2920.14679DOI Listing
October 2019

Ribosome dependence of persister cell formation and resuscitation.

J Microbiol 2019 Mar 26;57(3):213-219. Epub 2019 Feb 26.

Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania, 16802-4400, USA.

Since most bacterial cells are starving, they must enter a resting stage. Persister is the term used for metabolically-dormant cells that are not spores, and these cells arise from stress such as that from antibiotics as well as that from starvation. Because of their lack of metabolism, persister cells survive exposure to multiple stresses without undergoing genetic change; i.e., they have no inherited phenotype and behave as wild-type cells once the stress is removed and nutrients are presented. In contrast, mutations allow resistant bacteria to grow in the presence of antibiotics and slow growth allows tolerant cells to withstand higher concentrations of antibiotics; hence, there are three closely-related phenotypes: persistent, resistant, and tolerant. In addition, since dormancy is so prevalent, persister cells must have a means for resuscitating (since so many cells should obtain this resting state). In this review, we focus on what is known about the formation and resuscitation of persister cells.
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http://dx.doi.org/10.1007/s12275-019-8629-2DOI Listing
March 2019

Electron carriers increase electricity production in methane microbial fuel cells that reverse methanogenesis.

Biotechnol Biofuels 2018 25;11:211. Epub 2018 Jul 25.

1Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802-4400 USA.

Background: We previously reversed methanogenesis in microbial fuel cells (MFCs) to produce electricity for the first time from methane by combining an engineered archaeal strain that produces methyl-coenzyme M reductase from unculturable anaerobic methanotrophs (to capture methane and secrete acetate) with (to produce electrons from the generated acetate) and methane-acclimated sludge (to provide electron shuttles).

Results: Here, the power density in MFCs was increased 77-fold to 5216 mW/m and the current density in MFCs was increased 73-fold to 7.3 A/m by reducing the surface area of the cathode (to make reasonable comparisons to other MFCs), by changing the order the strains of the consortium were added to the anode compartment, and by adding additional electron carriers (e.g., humic acids and cytochrome C).

Conclusions: This power density and current density are comparable to the best for any MFC, including those with and spp. that utilize non-gaseous substrates. In addition, we demonstrate the methane MFC may be used to power a fan by storing the energy in a capacitor. Hence, MFCs that convert methane to electricity are limited by electron carriers.
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http://dx.doi.org/10.1186/s13068-018-1208-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058355PMC
July 2018

Single cell observations show persister cells wake based on ribosome content.

Environ Microbiol 2018 06 26;20(6):2085-2098. Epub 2018 Mar 26.

Department of Chemical Engineering, Pennsylvania State University, University Park, PA, 16802-4400, USA.

Since persister cells survive antibiotic treatments through dormancy and resuscitate to reconstitute infections, it is imperative to determine the rate at which these cells revive. Using two sets of Escherichia coli persister cells, those arising after antibiotic treatment at low levels and those generated at high levels by ceasing transcription via rifampicin pretreatment (shown to be bona fide persisters through eight sets of experiments), we used microscopy of single cells to determine that the resuscitation of dormant persisters is heterogeneous and includes cells that grow immediately. In all, five phenotypes were found during the observation of persister cells when fresh nutrients were added: (i) immediate division, (ii) immediate elongation followed by division, (iii) immediate elongation but no division, (iv) delayed elongation/division and (v) no growth. In addition, once cell division begins, the growth rate is that of exponential cells. Critically, the greater the ribosome content, the faster the persister cells resuscitate.
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http://dx.doi.org/10.1111/1462-2920.14093DOI Listing
June 2018

Viable but non-culturable and persistence describe the same bacterial stress state.

Environ Microbiol 2018 06 10;20(6):2038-2048. Epub 2018 Apr 10.

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, 16802-4400, USA.

Bacteria are often thought of as having two dormant phenotypes: the viable but non-culturable (VBNC) state and the persister state. Here we investigate the relatedness of the two stress-induced phenotypes at the single-cell level and examine cell morphology and quantify cell resuscitation. Using the classic starvation conditions to create VBNC cells, we found that the majority of the remaining Escherichia coli population are spherical, have empty cytosol and fail to resuscitate; however, some of the spherical cells resuscitate immediately (most probably those with dense cytosol). Critically, all the culturable cells in this starved population became persister cells within 14 days of starvation. We found that the persister cells initially are rod-like, have clear but limited membrane damage, can resuscitate immediately and gradually become spherical by aging. After 24 h, only rod-shaped persister cells survive, and all the spherical cells lyse. Both cell populations formed under the VBNC-inducing conditions and the persister conditions are metabolically inactive. Therefore, the bacterial population consists of dead cells and persister cells in the VBNC-inducing conditions; that is, the non-lysed particles that do not resuscitate are dead, and the dormant cells that resuscitate are persister cells. Hence, 'VBNC' and 'persister' describe the same dormant phenotype.
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http://dx.doi.org/10.1111/1462-2920.14075DOI Listing
June 2018

Current state and perspectives in hydrogen production by Escherichia coli: roles of hydrogenases in glucose or glycerol metabolism.

Appl Microbiol Biotechnol 2018 Mar 24;102(5):2041-2050. Epub 2018 Jan 24.

Department of Chemical Engineering, Pennsylvania State University, 43 Greenburg Complex, University Park, PA, 16802, USA.

Escherichia coli has been a robust host strain for much biological research, in particular, research in metabolic engineering, protein engineering, and heterologous gene expression. In this mini review, to understand bacterial hydrogen production by E. coli, the effect of glucose and glycerol metabolism on hydrogen production is compared, and the current approaches to enhance hydrogen production from glycerol as a substrate are reviewed. In addition, the argument from past to present on the functions of E. coli hydrogenases, hydrogenase 1, hydrogenase 2, hydrogenase 3, and hydrogenase 4 is summarized. Furthermore, based on the literature that the E. coli formate-hydrogen lyase is essential for bacterial hydrogen production via recombinant hydrogenases, research achievements from the past regarding heterologous production of hydrogenase are rethought.
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http://dx.doi.org/10.1007/s00253-018-8752-8DOI Listing
March 2018

Formation Mechanism of Flattened Top HFBI Domical Droplets.

J Phys Chem B 2016 04 12;120(15):3699-704. Epub 2016 Apr 12.

Advanced Catalytic Transformation Program for Carbon Utilization (ACT-C), Japan Science and Technology Agency (JST) , Tokyo 102-0076, Japan.

A water droplet assumes a spherical shape because of its own surface tension. However, water droplets containing dissolved hydrophobin (HFBI) have flat surfaces. In our previous study, the mechanism of this unique phenomenon was revealed. HFBI forms a self-organized membrane that has a densely packed and honeycomb-like structure. Furthermore, the buckling strength of the membrane is higher than the surface tension of the HFBI droplet. Therefore, an HFBI domical droplet has a flat surface. However, it was not clear why only the top of the domical droplet was flattened while other areas such as the side face were not. In this study, we observed HFBI domical droplets to investigate this phenomenon. The flat top area (self-organized HFBI membrane) remained parallel to the ground even if the substrate was tilted. Therefore, buoyancy was thought to be a factor affecting the HFBI membrane. In addition, the side face of the HFBI domical droplet was analyzed by atomic force microscopy and electrochemical impedance spectroscopy, and it was found that the sides of the HFBI droplet were not composed of densely packed HFBI membranes.
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http://dx.doi.org/10.1021/acs.jpcb.6b01306DOI Listing
April 2016

Flattened-Top Domical Water Drops Formed through Self-Organization of Hydrophobin Membranes: A Structural and Mechanistic Study Using Atomic Force Microscopy.

ACS Nano 2016 Jan 1;10(1):81-7. Epub 2015 Dec 1.

Advanced Catalytic Transformation Program for Carbon Utilization (ACT-C), Japan Science and Technology Agency (JST) , Tokyo 102-0076, Japan.

The Trichoderma reesei hydrophobin, HFBI, is a unique structural protein. This protein forms membranes by self-organization at air/water or water/solid interfaces. When HFBI forms a membrane at an air/water interface, the top of the water droplet is flattened. The mechanism underlying this phenomenon has not been explored. In this study, this unique phenomenon has been investigated. Self-organized HFBI membranes form a hexagonal structured membrane on the surface of water droplets; the structure was confirmed by atomic force microscopy (AFM) measurement. Assembled hexagons can form a planar sheet or a tube. Self-organized HFBI membranes on water droplets form a sheet with an array of hexagonal structures or a honeycomb structure. This membrane, with its arrayed hexagonal structures, has very high buckling strength. We hypothesized that the high buckling strength is the reason that water droplets containing HFBI form flattened domes. To test this hypothesis, the strength of the self-organized HFBI membranes was analyzed using AFM. The buckling strength of HFBI membranes was measured to be 66.9 mN/m. In contrast, the surface tension of water droplets containing dissolved HFBI is 42 mN/m. Thus, the buckling strength of a self-organized HFBI membrane is higher than the surface tension of water containing dissolved HFBI. This mechanistic study clarifies why the water droplets formed by self-organized HFBI membranes have a flattened top.
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http://dx.doi.org/10.1021/acsnano.5b04049DOI Listing
January 2016

Electrochemical properties of honeycomb-like structured HFBI self-organized membranes on HOPG electrodes.

Colloids Surf B Biointerfaces 2014 Nov 23;123:803-8. Epub 2014 Oct 23.

Department of Biological Functions Engineering, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Fukuoka, 808-0196, Japan; Research center for Eco-fitting Technology, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Fukuoka, 808-0196, Japan; Advanced Catalytic Transformation program for Carbon utilization (ACT-C), Japan Science and Technology Agency (JST), Tokyo, 102-0076, Japan. Electronic address:

HFBI (derived from Trichoderma sp.) is a unique structural protein, which forms a self-organized monolayer at both air/water interface and water/solid interfaces in accurate two-dimensional ordered structures. We have taken advantage of the unique functionality of HFBI as a molecular carrier for preparation of ordered molecular phase on solid substrate surfaces. The HFBI molecular carrier can easily form ordered structures; however, the dense molecular layers form an electrochemical barrier between the electrode and solution phase. In this study, the electrochemical properties of HFBI self-organized membrane-covered electrodes were investigated. Wild-type HFBI has balanced positive and negative charges on its surface. Highly oriented pyrolytic graphite (HOPG) electrodes coated with HFBI molecules were investigated electrochemically. To improve the electrochemical properties of this HFBI-coated electrode, the two types of HFBI variants, with oppositely charged surfaces, were prepared genetically. All three types of HFBI-coated HOPG electrode perform electron transfer between the electrode and solution phase through the dense HFBI molecular layer. This is because the HFBI self-organized membrane has a honeycomb-like structure, with penetrating holes. In the cases of HFBI variants, the oppositely charged HFBI membrane phases shown opposite electrochemical behaviors in electrochemical impedance spectroscopy. HFBI is a molecule with a unique structure, and can easily form honeycomb-like structures on solid material surfaces such as electrodes. The molecular membrane phase can be used for electrochemical molecular interfaces.
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http://dx.doi.org/10.1016/j.colsurfb.2014.10.018DOI Listing
November 2014

Solid-support immobilization of a "swing" fusion protein for enhanced glucose oxidase catalytic activity.

Colloids Surf B Biointerfaces 2013 Dec 2;112:186-91. Epub 2013 Aug 2.

Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Kitakyushu, Fukuoka, 808-0196, Japan; JST ACT-C, Japan.

The strategic surface immobilization of a protein can add new functionality to a solid substrate; however, protein activity, e.g., enzymatic activity, can be drastically decreased on immobilization onto a solid surface. The concept of a designed and optimized "molecular interface" is herein introduced in order to address this problem. In this study, molecular interface was designed and constructed with the aim of attaining high enzymatic activity of a solid-surface-immobilized a using the hydrophobin HFBI protein in conjunction with a fusion protein of HFBI attached to glucose oxidase (GOx). The ability of HFBI to form a self-organized membrane on a solid surface in addition to its adhesion properties makes it an ideal candidate for immobilization. The developed fusion protein was also able to form an organized membrane, and its structure and immobilized state on a solid surface were investigated using QCM-D measurements. This method of immobilization showed retention of high enzymatic activity and the ability to control the density of the immobilized enzyme. In this study, we demonstrated the importance of the design and construction of molecular interface for numerous purposes. This method of protein immobilization could be utilized for preparation of high throughput products requiring structurally ordered molecular interfaces, in addition to many other applications.
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http://dx.doi.org/10.1016/j.colsurfb.2013.07.051DOI Listing
December 2013
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