Trevor Douglas

Publications of Trevor Douglas

• A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose.

  Authors: Laura E Richert, Amy E Servid, Ann L Harmsen, Soo Han, James A Wiley, Trevor Douglas, Allen G Harmsen

  Vaccine. 03/2012;

  We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs).We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs). OVA-sHsp conjugation efficiency was dependent upon the stoichiometry and the length of the small molecule linker utilized, and the attachment position on the sHsp cage. When conjugated OVA-sHsp was delivered intranasally to naïve mice, the resulting immune response to OVA was accelerated and intensified, and OVA-specific IgG1 responses were apparent within 5 days after a single immunizing dose, illustrating its utility for vaccine development. If animals were pretreated with a disparate VLP, P22 (a non-replicative bacteriophage capsid), before OVA-sHsp conjugate immunization, OVA-specific IgG1 responses were apparent already by 4 days after a single immunizing dose of conjugate in OVA-naïve mice. Additionally, the mice pretreated with P22 produced high titer mucosal IgA, and isotype-switched OVA-specific serum IgG. Similarly, sHsp pretreatment enhanced the accumulation of lung germinal center B cells, T follicular helper cells, and increased polymeric Ig receptor expression, priming the lungs for subsequent IgG and IgA responses to influenza virus challenge. Thus, sHsp nanoparticles elicited quick and intense antibody responses and these accelerated responses could similarly be induced to antigen chemically conjugated to the sHsp. Pretreatment of mice with P22 further accelerated the onset of the antibody response to OVA-sHsp, demonstrating the utility of conjugating antigens to VLPs for pre-, or possibly post-exposure prophylaxis of lung, all without the need for adjuvant.

• RGD targeting of human ferritin iron-oxide nanoparticles enhances in vivo molecular MRI of experimental aortic aneurysms.

  Authors: Toshiro Kitagawa, Hisanori Kosuge, Masaki Uchida, Yasunori Iida, Ronald L Dalman, Trevor Douglas, Michael V McConnell

  Journal of cardiovascular magnetic resonance : official journal of the Society for Cardiovascular Magnetic Resonance. 02/2012; 14 Suppl 1:M9.

• Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection.

  Authors: Walid S Maaty, Kyla Selvig, Stephanie Ryder, Pavel Tarlykov, Jonathan K Hilmer, Joshua Heinemann, Joseph Steffens, Jamie C Snyder, Alice C Ortmann, Navid Movahed, Kevin Spicka, Lakshindra Chetia, Paul A Grieco, Edward A Dratz, Trevor Douglas, Mark J Young, Brian Bothner

  Journal of proteome research. 01/2012; 11(2):1420-32.

  Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for aWhere there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

• Characterization of the Bacteroides fragilis bfr gene product identifies a bacterial DPS-like protein and suggests evolutionary links in the ferritin superfamily.

  Authors: George H Gauss, Michael A Reott, Edson R Rocha, Mark J Young, Trevor Douglas, C Jeffrey Smith, C Martin Lawrence

  Journal of bacteriology. 01/2012; 194(1):15-27.

  A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allowsA factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacterium's extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O(2) or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer.

• Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer.

  Authors: Oleg A Zadvornyy, Janice E Lucon, Robin Gerlach, Nikolay A Zorin, Trevor Douglas, Timothy E Elgren, John W Peters

  Journal of inorganic biochemistry. 01/2012; 106(1):151-5.

  The potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the developmentThe potential of hydrogen as a clean renewable fuel source and the finite reserves of platinum metal to be utilized in hydrogen production catalysts have provided the motivation for the development of non-noble metal-based solutions for catalytic hydrogen production. There are a number of microorganisms that possess highly efficient hydrogen production catalysts termed hydrogenases that generate hydrogen under certain metabolic conditions. Although hydrogenases occur in photosynthetic microorganisms, the oxygen sensitivity of these enzymes represents a significant barrier in directly coupling hydrogen production to oxygenic photosynthesis. To overcome this barrier, there has been considerable interest in identifying or engineering oxygen tolerant hydrogenases or generating mimetic systems that do not rely on oxygen producing photocatalysts. In this work, we demonstrate photo-induced hydrogen production from a stable [NiFe]-hydrogenase coupled to a [Ru(2,2'-bipyridine)(2)(5-amino-1,10-phenanthroline)](2+) photocatalyst. When the Ru(II) complex is covalently attached to the hydrogenase, photocatalytic hydrogen production occurs more efficiently in the presence of a redox mediator than if the Ru(II) complex is simply present in solution. Furthermore, sustained hydrogen production occurs even in the presence of oxygen by presumably creating a local anoxic environment through the reduction of oxygen similar to what is proposed for oxygen tolerant hydrogenases. These results provide a strong proof of concept for engineering photocatalytic hydrogen production in the presence of oxygen using biohybrid mimetic systems.

• Topological biosignatures: large-scale structure of chemical networks from biology and astrochemistry.

  Authors: Craig Jolley, Trevor Douglas

  Astrobiology. 12/2011; 12(1):29-39.

  The chemical basis of life involves more than simply the presence of biological molecules; biochemical systems embody a complex network of reactions with characteristic topological features. At theThe chemical basis of life involves more than simply the presence of biological molecules; biochemical systems embody a complex network of reactions with characteristic topological features. At the same time, chemical complexity is also present in nonbiological contexts, inviting us to clarify the relationship between chemistry and life through comparative studies. This study examines chemical networks from biology (the metabolism of E. coli) and astronomy (gas-phase reactions in dark molecular clouds) to establish common topological features that may be generic for any complex chemical system, as well as clear differences that may be topological signatures of life. The biological and astrochemical networks exhibit different scaling behaviors, and the network motifs found in the two systems show similarities as well as significant differences. The PageRank algorithm was used to quantify the degree to which individual species act primarily as products or reactants; in the metabolic network, these two roles are nearly identical for most species, whereas the astrochemical network shows a clearer partitioning into reactants and products.

• Site-directed coordination chemistry with P22 virus-like particles.

  Authors: Masaki Uchida, David S Morris, Sebyung Kang, Craig C Jolley, Janice Lucon, Lars O Liepold, Ben LaFrance, Peter E Prevelige, Trevor Douglas

  Langmuir : the ACS journal of surfaces and colloids. 12/2011; 28(4):1998-2006.

  Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have beenProtein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.

• Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22.

  Authors: Alison O'Neil, Courtney Reichhardt, Benjamin Johnson, Peter E Prevelige, Trevor Douglas

  Angewandte Chemie (International ed. in English). 06/2011; 50(32):7425-8.

• Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV).

  Authors: Jennifer Fulton Wirth, Jamie C Snyder, Rebecca A Hochstein, Alice C Ortmann, Deborah A Willits, Trevor Douglas, Mark J Young

  Virology. 06/2011; 415(1):6-11.

  Our understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virusOur understanding of archaeal viruses has been limited by the lack of genetic systems for examining viral function. We describe the construction of an infectious clone for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). STIV was isolated from a high temperature (82°C) acidic (pH 2.2) hot spring in Yellowstone National Park and replicates in the archaeal model organism Sulfolobus solfataricus (Rice et al., 2004). While STIV is one of most studied archaeal viruses, little is known about its replication cycle. The development of an STIV infectious clone allows for directed gene disruptions and detailed genetic analysis of the virus. The utility of the STIV infectious clone was demonstrated by gene disruption of STIV open reading frame (ORF) B116 which resulted in crippled virus replication, while disruption of ORFs A197, C381 and B345 was lethal for virus replication.

• Templated assembly of organic-inorganic materials using the core shell structure of the P22 bacteriophage.

  Authors: Courtney Reichhardt, Masaki Uchida, Alison O'Neil, Rui Li, Peter E Prevelige, Trevor Douglas

  Chemical communications (Cambridge, England). 06/2011; 47(22):6326-8.

  Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure,Biomimetic chemistry offers new approaches to supramolecular materials synthesis and assembly. We have demonstrated that an assembled viral protein cage, comprising an organic core-shell structure, can be used as a template for the size constrained synthesis of Fe(2)O(3). Particle nucleation is directed by the inner scaffold protein layer, while the size constraints are determined by the outer capsid layer.

• RGD-Conjugated Human Ferritin Nanoparticles for Imaging Vascular Inflammation and Angiogenesis in Experimental Carotid and Aortic Disease.

  Authors: Toshiro Kitagawa, Hisanori Kosuge, Masaki Uchida, Monica M Dua, Yasunori Iida, Ronald L Dalman, Trevor Douglas, Michael V McConnell

  Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging. 06/2011;

  PURPOSE: Inflammation and angiogenesis are important contributors to vascular disease. We evaluated imaging both of these biological processes, using Arg-Gly-Asp (RGD)-conjugated human ferritinPURPOSE: Inflammation and angiogenesis are important contributors to vascular disease. We evaluated imaging both of these biological processes, using Arg-Gly-Asp (RGD)-conjugated human ferritin nanoparticles (HFn), in experimental carotid and abdominal aortic aneurysm (AAA) disease. PROCEDURES: Macrophage-rich carotid lesions were induced by ligation in hyperlipidemic and diabetic FVB mice (n = 16). AAAs were induced by angiotensin II infusion in apoE(-/-) mice (n=10). HFn, with or without RGD peptide, was labeled with Cy5.5 and injected intravenously for near-infrared fluorescence imaging. RESULTS: RGD-HFn showed significantly higher signal than HFn in diseased carotids and AAAs relative to non-diseased regions, both in situ (carotid: 1.88 ± 0.30 vs. 1.17 ± 0.10, p = 0.04; AAA: 2.59 ± 0.24 vs. 1.82 ± 0.16, p = 0.03) and ex vivo. Histology showed RGD-HFn colocalized with macrophages in carotids and both macrophages and neoangiogenesis in AAA lesions. CONCLUSIONS: RGD-HFn enhances vascular molecular imaging by targeting both vascular inflammation and angiogenesis, and allows more comprehensive detection of high-risk atherosclerotic and aneurysmal vascular diseases.

• Monitoring structural transitions in icosahedral virus protein cages by site-directed spin labeling.

  Authors: Robert J Usselman, Eric D Walter, Debbie Willits, Trevor Douglas, Mark Young, David J Singel

  Journal of the American Chemical Society. 03/2011; 133(12):4156-9.

  This work describes an approach for calculating and measuring dipolar interactions in multispin systems to monitor conformational changes in icosahedral protein cages using site-directed spinThis work describes an approach for calculating and measuring dipolar interactions in multispin systems to monitor conformational changes in icosahedral protein cages using site-directed spin labeling. Cowpea chlorotic mottle virus (CCMV) is used as a template that undergoes a pH-dependent reversible capsid expansion wherein the protein cage swells by 10%. The sequence-position-dependent geometric presentation of attached spin-label groups provides a strategy for targeting amino acid residues most probative of structural change. The labeled protein cage residues and structural transition were found to affect the local mobility and dipolar interactions of the spin label, respectively. Line-shape changes provided a spectral signature that could be used to follow the conformational change in CCMV coat dynamics. The results provide evidence for a concerted swelling process in which the cages exist in only two structural forms, with essentially no intermediates. This methodology can be generalized for all symmetry types of icosahedral protein architectures to monitor protein cage dynamics.

• Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions.

  Authors: Masaki Uchida, Hisanori Kosuge, Masahiro Terashima, Deborah A Willits, Lars O Liepold, Mark J Young, Michael V McConnell, Trevor Douglas

  ACS nano. 03/2011; 5(4):2493-502.

  Cage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heatCage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nanoscale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.

• A water-soluble carbon nanotube network conjugated by nanoparticles with defined nanometre gaps.

  Authors: Mime Kobayashi, Shinya Kumagai, Bin Zheng, Yukiharu Uraoka, Trevor Douglas, Ichiro Yamashita

  Chemical communications (Cambridge, England). 02/2011; 47(12):3475-7.

  Cage-shaped proteins with an affinity for carbonaceous materials were constructed and used to assemble a nanostructure in which single-walled carbon nanotubes are surrounded by cobalt oxideCage-shaped proteins with an affinity for carbonaceous materials were constructed and used to assemble a nanostructure in which single-walled carbon nanotubes are surrounded by cobalt oxide nanoparticles with nanometre gaps. By changing the size of proteins and materials incorporated inside the cavity, similar structures with distinctively different properties can be fabricated.

• RGD targeting of human ferritin iron oxide nanoparticles can enhance in vivo carotid MRI of experimental atherosclerosis

  Authors: Toshiro Kitagawa, Hisanori Kosuge, Masaki Uchida, Trevor Douglas, Michael McConnell

  Journal of Cardiovascular Magnetic Resonance. 01/2011;

• Gd-based protein cage nanoparticles provide enhanced r1 relaxivity and detect experimental atherosclerosis

  Authors: Lars Liepold, Masaki Uchida, Abedin Md Joynald, Shefah Qazi, Hisanori Kosuge, Toshiro Kitagawa, Michael McConnell, Trevor Douglas

  Journal of Cardiovascular Magnetic Resonance. 01/2011;

• Structure and photoelectrochemistry of a virus capsid-TiO2 nanocomposite.

  Authors: Craig Jolley, Michael Klem, Richard Harrington, John Parise, Trevor Douglas

  Nanoscale. 12/2010; 3(3):1004-7.

  The use of protein templates to direct the formation of inorganic nanostructures offers a novel bio-inspired route to nanomaterial synthesis that avoids the use of harsh reaction conditions andThe use of protein templates to direct the formation of inorganic nanostructures offers a novel bio-inspired route to nanomaterial synthesis that avoids the use of harsh reaction conditions and offers unique functionalities including biocompatibility and hierarchical assembly. Pair distribution function (PDF) analysis from total X-ray scattering has been used to determine the structure of TiO2 nanoparticles grown within an icosahedral virus capsid. The protein-TiO2 composites are similar to nanocrystalline anatase and show photocatalytic activity. PDF analysis is ideally suited to the study of protein-inorganic nanocomposites, and may be able to provide information about the hard/soft interface.

• Human ferritin cages for imaging vascular macrophages.

  Authors: Masahiro Terashima, Masaki Uchida, Hisanori Kosuge, Philip S Tsao, Mark J Young, Steven M Conolly, Trevor Douglas, Michael V McConnell

  Biomaterials. 11/2010; 32(5):1430-7.

  Atherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, anAtherosclerosis is a leading cause of death worldwide. Macrophages are key components of vascular inflammation, which contributes to the development and complications of atherosclerosis. Ferritin, an iron storage and transport protein, has been found to accumulate in macrophages in human atherosclerotic plaques. We hypothesized that ferritin could serve as an intrinsic nano-platform to target delivery of imaging agents to vascular macrophages to detect high-risk atherosclerotic plaques. Here we show that engineered human ferritin protein cages, either conjugated to the fluorescent Cy5.5 molecule or encapsulating a magnetite nanoparticle, are taken up in vivo by macrophages in murine atherosclerotic carotid arteries and can be imaged by fluorescence and magnetic resonance imaging. These results indicate that human ferritin can serve as a nanoparticle platform to image vascular inflammation in vivo.

• Ion accumulation in a protein nanocage: finding noisy temporal sequences using a genetic algorithm.

  Authors: Craig C Jolley, Trevor Douglas

  Biophysical journal. 11/2010; 99(10):3385-93.

  Many pathogenic bacteria are able to survive attack by the host's immune system because of antioxidant systems that mitigate the effects of reactive oxygen species. Dps is a hollow 12-subunit proteinMany pathogenic bacteria are able to survive attack by the host's immune system because of antioxidant systems that mitigate the effects of reactive oxygen species. Dps is a hollow 12-subunit protein nanocage that prevents oxidative damage by oxidizing and sequestering intracellular Fe(2+); the resulting Fe(3+) forms an iron oxyhydroxide nanoparticle in the cage interior. Charged sites on the protein nanocage create an electrostatic gradient that guides ions through well-defined pores that connect the cage interior with the surrounding solution and toward nucleation sites on the cage interior. In this study, we use all-atom molecular dynamics to simulate the motion of simple cations into the dodecameric cage formed by the Dps protein from Listeria monocytogenes. Ion trajectories are analyzed by using a novel, to our knowledge, genetic algorithm to determine the temporal sequence of ion-protein interactions. Ions enter Dps through well-defined pores at the ferritinlike C(3) axes, with negatively-charged residues on the outside of the cage forming a fairly well-defined entrance pathway. This method of trajectory analysis may be broadly applicable in situations where the spatial localization of ions or other small molecules is electrostatically driven by a biomolecule.

• Implementation of p22 viral capsids as nanoplatforms.

  Authors: Sebyung Kang, Masaki Uchida, Alison O'Neil, Rui Li, Peter E Prevelige, Trevor Douglas

  Biomacromolecules. 10/2010; 11(10):2804-9.

  Viral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structuralViral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structural flexibility of viral capsids and generated two morphologically different types of viral nanoplatforms from the bacteriophage P22 capsids. Their interior surfaces were genetically manipulated for site-specific attachment of a biotin linker. The extent of internal modifications in each capsid form was characterized by high-resolution mass spectrometry and the analyses revealed that the reactivity of the genetically introduced residues located on the internal surface changes according to the structural transformation of the capsid. Internally modified capsids having 10 nm diameter pores at the 12 icosahedral vertices, so-called wiffle-balls (WB), exhibited the capability to entrap the large tetrameric protein complex streptavidin via the biotin linker anchored onto the interior surface of the WB.