Browsing by Author "Douglas, Trevor"

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Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo
(2007-12) Kaiser, Coleen R.; Flenniken, Michelle L.; Gillitzer, Eric; Harmsen, Ann L.; Harmsen, Allen G.; Jutila, Mark A.; Douglas, Trevor; Young, Mark J.
Protein cage nanoparticles have the potential to serve as multifunctional cell targeted, imaging and therapeutic platforms for broad applications in medicine. However, before they find applications in medicine, their biocompatibility in vivo needs to be demonstrated. We provide here baseline biodistribution information of two different spherical protein cage nanoplatforms, the 28 nm viral Cowpea chlorotic mottle virus (CCMV) and the 12 nm heat shock protein (Hsp) cage. In naïve and immunized mice both nanoplatforms show similar broad distribution and movement throughout most tissues and organs, rapid excretion, the absence of long term persistence within mice tissue and organs, and no overt toxicity after a single injection. These results suggest that protein cage based nanoparticles may serve as safe, biocompatible, nanoplatforms for applications in medicine.
Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation
(2018-06) Kant, Ravi; Llauro, Aida; Rayaprolu, Vamseedhar; Qazi, Shefah; de Pablo, Pedro J.; Douglas, Trevor; Bothner, Brian
The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.
Controlled Modular Multivalent Presentation of the CD40 Ligand on P22 Virus-like Particles Leads to Tunable Amplification of CD40 Signaling
(American Chemical Society, 2021-11) Goodall, Cheri Peyton; Schwartz, Benjamin; Selivanovitch, Ekaterina; Avera, John; Wang, Joseph; Miettinen, Heini; Douglas, Trevor
Ligands of the tumor necrosis factor superfamily (TNFSF) are appealing targets for immunotherapy research due to their integral involvement in stimulation or restriction of immune responses. TNFSF-targeted therapies are currently being developed to combat immunologically based diseases and cancer. A crucial determinant of effective TNFSF receptor binding and signaling is the trimeric quaternary structure of the ligand. Additionally, ligand multivalency is essential to propagate strong signaling in effector cells. Thus, designing a synthetic platform to display trimeric TNFSF ligands in a multivalent manner is necessary to further the understanding of ligand–receptor interactions. Viral nanocages have architectures that are amenable to genetic and chemical modifications of both their interior and exterior surfaces. Notably, the exterior surface of virus-like particles can be utilized as a platform for the modular multivalent presentation of target proteins. In this study, we build on previous efforts exploring the bacteriophage P22 virus-like particle for the exterior multivalent modular display of a potent immune-stimulating TNFSF protein, CD40 ligand (CD40L). Using a cell-based reporter system, we quantify the effects of tunable avidity on CD40 signaling by CD40L displayed on the surface of P22 nanocages. Multivalent presentation of CD40L resulted in a 53.6-fold decrease of the half maximal effective concentration (EC50) compared to free CD40L, indicating higher potency. Our results emphasize the power of using P22-based biomimetics to study ligand−receptor interactions within their proper structural context, which may contribute to the development of effective immune modulators.
Enzyme encapsulation in the P22 viral capsid as a platform for biological nanoreactors
(2013-03) Schwarz, Benjamin; Douglas, Trevor
The capsid of the Salmonella typhimurium bacteriophage P22 presents a platform for encapsidation of protein cargo through genetic fusion. The capsid spontaneously assembles via interactions between a scaffold protein and the interior of the coat protein shell. By fusing proteins to a truncated scaffold protein it is possible to package cargo inside the capsid which has potential to generate thermostable, dynamic, targetable nanoreactors. The present work proves this concept with the encapsidation of two thermophilic enzymes, the monomeric alcohol dehydrogenase AdhD and the tetrameric β;-glucosidase CelB both from Pyrococcus furiousus. Both enzymes are shown to be active in the packaged form and present within the capsid in large numbers, 85 monomers /capsid in CelB and 250 monomers/capsid in AdhD. The functionalized capsids were shown to retain the temperature dependent maturation seen in the wild type.
High-density targeting of a viral multifunctional nanoplatform to a pathogenic, biofilm-forming bacterium
(2007-04) Suci, Peter A.; Berglund, Deborah L.; Liepold, Lars Otto; Brumfield, Susan; Pitts, Betsey; Davison, William M.; Oltrogge, Luke; Hoyt, Kathryn Olivia; Codd, Sarah L.; Stewart, Philip S.; Young, Mark J.; Douglas, Trevor
Nanomedicine directed at diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. Here, we targeted a spherical, icosahedral viral nanoplatform to a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding mediated through specific protein-ligand interactions exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto cells. The fluorescence-imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.
A Human ferritin iron oxide nano-composite magnetic resonance contrast agent
(2008-11) Uchida, M.; Terashima, Masahiro; Cunningham, Charles H.; Suzuki, Yoriyasu; Willits, Deborah Ann; Willis, Ann F.; Yang, Philip C.; Tsao, Philip S.; McConnell, Michael V.; Young, Mark J.; Douglas, Trevor
Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)-iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R2 relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn-iron oxide contrast agent to assess inflammatory disease status such as macrophage-rich atherosclerotic plaques in vivo.
Incorporation of HNP-1 into a Protein Cage Architecture Based on the Bacteriophage P22
(2013-03) Wheeler, Luke; Douglas, Trevor
Utilizing molecular biology techniques, a gene construct was created that encodes for a modified protein cage architecture based on the bacteriophage p22. This specific protein cage has a genetic fusion to the scaffolding protein (which is necessary for particle assembly) which results in the incorporation of the antibacterial and antifungal Human Alpha Defensin 1, also known as Human Neutrophil Peptide-1 (HNP-1) on the inside of the protein cage, with one HNP-1 attached to each subunit. This protein was expressed in E. coli expression cells (BL-21 line), isolated and purified. The identity of the modified protein cage architecture was confirmed using a combination of mass spectrometry, SDS PAGE, dynamic light scattering, multi-angle light scattering and agarose gel electrophoresis. Preliminary bioactivity assays using aspergillus fumigatus indicate that the protein construct does not have antifungal activity and instead increases the metabolic rate of treated fungi. This, coupled with the high yield of protein isolated from e. coli, indicates that the containment of the antibacterial peptide within the P22 protein cage results in the shielding of exposed organisms from the cytotoxic activity of the enclosed HNP-1.
Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses
(2009-09) Wiley, James A.; Richert, Laura E.; Swain, Steve D.; Harmsen, Ann L.; Barnard, Dale L.; Randall, Troy D.; Jutila, Mark A.; Douglas, Trevor; Broomell, Chris; Young, Mark J.; Harmsen, Allen G.
Background Destruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage. Methodology/Principal Findings Mice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms. Conclusions/Significance The immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.
Intracellular distribution of macrophage targeting ferritin–iron oxide nanocomposite
(2009-01) Uchida, M.; Willits, Deborah Ann; Muller, Karin; Willis, Ann F.; Jackiw, Larissa; Jutila, Mark A.; Young, Mark J.; Porter, Alexandra E.; Douglas, Trevor
Intracellular distribution of iron oxide nanoparticlesincorporated within a ferritin mutant that displays genetically introduced cell-targeted peptides (RGD-4C) on its exterior surface are investigated using scanning transmission electron microscopy with a high-angle annular dark-field detector. The particles (indicated by arrows) internalized into macrophages much more effectively than those with noncell-targeted ferritin.
Melanoma and Lymphocyte Cell Specific Targeting Incorporated into a Heat Shock Protein Cage Architecture
(2006) Flenniken, Michelle L.; Willits, Deborah A.; Harmsen, Ann L.; Liepold, Lars O.; Harmsen, Allen G.; Young, Mark J.; Douglas, Trevor
Protein cages, including viral capsids, ferritins, and heat shock proteins (Hsps), can serve as nanocontainers for biomedical applications. They are genetically and chemically malleable platforms, with potential as therapeutic and imaging agent delivery systems. Here, both genetic and chemical strategies were used to impart cell-specific targeting to the Hsp cage from Methanococcus jannaschii. A tumor vasculature targeting peptide was incorporated onto the exterior surface of the Hsp cage. This protein cage bound to αvβ3 integrin-expressing cells. Cellular tropism was also imparted by conjugating anti-CD4 antibodies to the exterior of Hsp cages. These Ab-Hsp cage conjugates specifically bound to CD4+ cells. Protein cages have the potential to simultaneously incorporate multiple functionalities, including cell-specific targeting, imaging, and therapeutic agent delivery. We demonstrate the simultaneous incorporation of two functionalities, imaging and cell-specific targeting, onto the Hsp protein cage.
Microbe manufacturers of semiconductors
(2004-11) Flenniken, Michelle L.; Allen, Mark; Douglas, Trevor
Synthesis of cadmium sulfide (CdS) semiconductor nanoparticles within a prokaryotic organism is reported for the first time by Sweeney et al. [1]. This paper demonstrates the utility of microorganisms to perform chemistries outside the scope of their “normal” metabolism and offers an environmentally benign synthesis of CdS nanoparticles.
Photo-induced H2 production by [NiFe]-hydrogenase from T. roseopersicina covalently linked to a Ru(II) photosensitizer
(2012-01) Zadvornyy, Oleg A.; Lucon, Janice E.; Gerlach, Robin; Zorin, Nikolay A.; Douglas, Trevor; Elgren, T. E.; Peters, John W.
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 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-amino1,10 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.
Something old, something new, something borrowed; how the thermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress
(2009-09) Maaty, Walid S.; Wiedenheft, Blake A.; Tarlykov, Pavel V.; Schaff, Nathan; Heinemann, Joshua V.; Robison-Cox, James; Dougherty, Amanda; Blum, Paul; Lawrence, C. Martin; Douglas, Trevor; Young, Mark J.; Bothner, Brian
To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H2O2) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 µM H2O2. Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had significant changes in abundance. A recently characterized ferritin-like antioxidant protein, DPSL, was the most highly regulated species of mRNA and protein, in addition to being post-translationally modified. As expected, a number of antioxidant related mRNAs and proteins were differentially regulated. Three of these, DPSL, superoxide dismutase, and peroxiredoxin were shown to interact and likely form a novel supramolecular complex for mitigating oxidative damage. A scheme for the ability of this complex to perform multi-step reactions is presented. Despite the central role played by DPSL, cells maintained a lower level of protection after disruption of the dpsl gene, indicating a level of redundancy in the oxidative stress pathways of S. solfataricus. This work provides the first “omics” scale assessment of the oxidative stress response for an archeal organism and together with a network analysis using data from previous studies on bacteria and eukaryotes reveals evolutionarily conserved pathways where complex and overlapping defense mechanisms protect against oxygen toxicity.
Targeted delivery of a photosensitizer to Aggregatibacter actinomycetemcomitans biofilm
(2010-04) Suci, Peter A.; Kang, Sebyung; Gmür, Rudolf; Douglas, Trevor; Young, Mark J.
The ability to selectively target specific biofilm species with antimicrobials would enable control over biofilm consortium composition, with medical applications in treatment of infections on mucosal surfaces that are colonized by a mixture of beneficial and pathogenic microorganisms. We functionalized a genetically engineered multimeric protein with both a targeting moiety (biotin) and either a fluorophore or a photosensitizer (SnCe6). Biofilm microcolonies of Aggregatibacter actinomycetemcomitans, a periodontal pathogen, were targeted with the multifunctional dodecamer. Streptavidin was used to couple biotinylated dodecamer to a biotinylated anti-A. actinomycetemcomitans antibody. This modular targeting approach enabled us to increase the loading of photosensitizer onto the cells by a cycle of amplification. Scanning laser confocal microscopy was used to characterize transport of fluorescently tagged dodecamer into the microcolonies and targeting of the cells with biotin-labeled, fluorescently tagged dodecamer. Light-induced activity of the targeted photosensitizer reduced the viability of A. actinomycetemcomitans biofilm, as indicated by membrane permeability to propidium iodide. The functionalized multimeric protein promises to be a useful tool for controlling periodontal biofilm consortia and offers a modular design whereby moieties that target different species can be readily combined with the functionalized protein construct.
Virus-like Particle-Induced Protection against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function
(2012-07) Rynda-Apple, Agnieszka; Dobrinen, Erin; McAlpine, Mark; Read, Amanda; Harmsen, Ann L.; Richert, Laura E.; Calverley, Matthew; Pallister, Kyler; Voyich, Jovanka M.; Wiley, James A.; Johnson, Ben; Young, Mark J.; Douglas, Trevor; Harmsen, Allen G.
The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms.