[Show abstract][Hide abstract] ABSTRACT: Limited by culture-dependent methods the number of viruses identified from thermophilic Archaea and Bacteria is still very small. In this study we retrieved viral sequences from six hot spring metagenomes isolated worldwide, revealing a wide distribution of four archaeal viral families, Ampullaviridae, Bicaudaviridae, Lipothrixviridae and Rudiviridae. Importantly, we identified ten complete or near complete viral genomes allowing, for the first time, an assessment of genome conservation and evolution of the Ampullaviridae family as well as Sulfolobus Monocaudavirus 1 (SMV1) related viruses. Among the novel genomes, one belongs to a putative thermophilic virus infecting the bacterium Hydrogenobaculum, for which no virus has been reported in the literature. Moreover, a high viral diversity was observed in the metagenomes, especially among the Lipothrixviridae, as indicated by the large number of unique contigs and the lack of a completely assembled genome for this family. This is further supported by the large number of novel genes in the complete and partial genomes showing no sequence similarities to public databases. CRISPR analysis revealed hundreds of novel CRISPR loci and thousands of novel CRISPR spacers from each metagenome, reinforcing the notion of high viral diversity in the thermal environment.
Full-text · Article · Oct 2015 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Hot springs are natural habitats for thermophilic Archaea and Bacteria. In this paper, we present the metagenomic analysis of eight globally distributed terrestrial hot springs from China, Iceland, Italy, Russia, and the USA with a temperature range between 61 and 92 ∘C and pH between 1.8 and 7. A comparison of the biodiversity and community composition generally showed a decrease in biodiversity with increasing temperature and decreasing pH. Another important factor shaping microbial diversity of the studied sites was the abundance of organic substrates. Several species of the Crenarchaeal order Thermoprotei were detected, whereas no single bacterial species was found in all samples, suggesting a better adaptation of certain archaeal species to different thermophilic environments. Two hot springs show high abundance of Acidithiobacillus, supporting the idea of a true thermophilic Acidithiobacillus species that can thrive in hyperthermophilic environments. Depending on the sample, up to 58 % of sequencing reads could not be assigned to a known phylum, reinforcing the fact that a large number of microorganisms in nature, including those thriving in hot environments remain to be isolated and characterized.
Full-text · Article · Feb 2015 · Microbial Ecology
[Show abstract][Hide abstract] ABSTRACT: A pH-sensitive ciprofloxacin prodrug was synthesized and targeted against biofilms of the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). The dose required to reduce the viability of a mature biofilm of Aa by ∼80% was in the range of ng cm(-2) of colonized area (mean biofilm density 2.33 × 10(9) cells cm(-2)). A mathematical model was formulated that predicts the temporal change in the concentration of ciprofloxacin in the Aa biofilm as the drug is released and diffuses into the bulk medium. The predictions of the model were consistent with the extent of killing obtained. The results demonstrate the feasibility of the strategy to induce mortality, and together with the mathematical model, provide the basis for design of targeted antimicrobial prodrugs for the topical treatment of oral infections such as periodontitis. The targeted prodrug approach offers the possibility of optimizing the dose of available antimicrobials in order to kill a chosen pathogen while leaving the commensal microbiota relatively undisturbed.
[Show abstract][Hide abstract] ABSTRACT: Mucosal surfaces such as the gut, vagina and oral cavity are colonized by microbiota that are an integral component of the healthy ecosystem. Recent molecular techniques make it feasible to correlate antimicrobial dosing levels with changes in microbiome composition. The objective of this study was to characterize the rat oral plaque microbiome composition at doses of ciprofloxacin that were considerably above and below nominal in vitro minimal inhibitory concentrations (MICs) of a variety of gram positive oral commensal bacteria. We exposed the oral cavities of rats to relatively low (0.1 μg/mL) and high (20 μg/mL) doses of ciprofloxacin in the drinking water over a 3 day period. Plaque microbiota were characterized using 454 pyrosequencing. The rat indigenous community was dominated by Rothia (74.4 %) and Streptococcus genera (4.7%). Dosing at 0.1 μg/mL was associated with changes in Rothia and Streptococcus genera which were not significant, while dosing at 20 μg/mL caused pronounced (significant) reduction in the relative abundance of the Streptococcus genus. Taxonomic independent analysis indicated that the perturbation in the overall community structure attributed to dosing with ciprofloxacin at either the low or high dose was relatively low. The results suggest that it is feasible to use an antimicrobial dosing regime to selectively target a specific subset of a mucosal microbiome for elimination with minimal perturbation of the entire community.
[Show abstract][Hide abstract] ABSTRACT: Electron magnetic resonance (EMR) spectroscopy was used to determine the magnetic properties of maghemite (γ-Fe(2)O(3)) nanoparticles formed within size-constraining Listeria innocua (LDps)-(DNA-binding protein from starved cells) protein cages that have an inner diameter of 5 nm. Variable-temperature X-band EMR spectra exhibited broad asymmetric resonances with a superimposed narrow peak at a gyromagnetic factor of g ≈ 2. The resonance structure, which depends on both superparamagnetic fluctuations and inhomogeneous broadening, changes dramatically as a function of temperature, and the overall linewidth becomes narrower with increasing temperature. Here, we compare two different models to simulate temperature-dependent lineshape trends. The temperature dependence for both models is derived from a Langevin behavior of the linewidth resulting from "anisotropy melting." The first uses either a truncated log-normal distribution of particle sizes or a bi-modal distribution and then a Landau-Liftshitz lineshape to describe the nanoparticle resonances. The essential feature of this model is that small particles have narrow linewidths and account for the g ≈ 2 feature with a constant resonance field, whereas larger particles have broad linewidths and undergo a shift in resonance field. The second model assumes uniform particles with a diameter around 4 nm and a random distribution of uniaxial anisotropy axes. This model uses a more precise calculation of the linewidth due to superparamagnetic fluctuations and a random distribution of anisotropies. Sharp features in the spectrum near g ≈ 2 are qualitatively predicted at high temperatures. Both models can account for many features of the observed spectra, although each has deficiencies. The first model leads to a nonphysical increase in magnetic moment as the temperature is increased if a log normal distribution of particles sizes is used. Introducing a bi-modal distribution of particle sizes resolves the unphysical increase in moment with temperature. The second model predicts low-temperature spectra that differ significantly from the observed spectra. The anisotropy energy density K(1), determined by fitting the temperature-dependent linewidths, was ∼50 kJ/m(3), which is considerably larger than that of bulk maghemite. The work presented here indicates that the magnetic properties of these size-constrained nanoparticles and more generally metal oxide nanoparticles with diameters d < 5 nm are complex and that currently existing models are not sufficient for determining their magnetic resonance signatures.
Full-text · Article · Oct 2012 · Journal of Applied Physics
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · May 2012 · American Journal Of Pathology
[Show abstract][Hide abstract] ABSTRACT: There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.
Full-text · Article · Feb 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Introduction Protein Cage Based Magnetic Materials as MRI Contrast Agents Protein Cages as T1 Agents Protein Cages as T2 MRI Contrast Agents Protein Cages as Contrast Agents for Imaging Atherosclerotic Plaques Protein Cages as Vehicles for Targeted Delivery of Imaging Agents to Biofilm Infections Conclusion References
[Show abstract][Hide abstract] ABSTRACT: Periodontal disease is associated with a pathogen-induced transition to a chronic destructive inflammatory response. Since commensals may either passively or actively contribute to immune homeostasis, therapies aimed at selectively reducing the competitive advantage of pathogens may be effective supplements to traditional methods. We developed an in vitro system to grow biofilms composed of the pathogen (Aggregatibacter actinomycetemcomitans) and the commensal (Streptococcus sanguinis). We used the biofilm model to determine the feasibility of selectively killing the pathogen using the fluoroquinolone, ciprofloxacin.
Biofilms were exposed to relevant ciprofloxacin doses during the first 24h of development, with subsequent removal of the ciprofloxacin for a 24h period. Biofilm growth was assessed by confocal laser scanning microscopy, crystal violet staining and DNA abundance.
Exposure to 0.01mg/L or 0.5mg/L ciprofloxacin significantly reduced the microcolony size and cell surface density of A. actinomycetemcomitans in the dual species biofilm over a 24h period whilst allowing uninhibited S. sanguinis biofilm formation. A. actinomycetemcomitans biofilm development was insignificant over a subsequent 24h period after removal of the ciprofloxacin indicating that A. actinomycetemcomitans cells were killed.
A. actinomycetemcomitans residing in a dual species biofilm with the commensal, S. sanguinis can be selectively killed, or at least rendered metabolically inactive, by treatment with ciprofloxacin. The dual species biofilm model will be a useful tool for designing in vivo studies to determine the efficacy of selective killing agents as an adjunct treatment of localized aggressive forms of periodontal disease.
No preview · Article · Apr 2011 · Archives of oral biology
[Show abstract][Hide abstract] ABSTRACT: 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 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.
Full-text · Article · Mar 2011 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Coordination polymers (CPs) have garnered much attention in the past several years for the relative facility of their synthesis and potential benefit in diverse applications such as gas separation, energy storage, drug delivery and as novel bio-imaging compounds. To date there have been relatively few reports of CPassembly from peptide-based precursors. CPs generated from biomolecules offer several potential advantages over their synthetic counterparts including extensive structural diversity, intrinsic chirality and the capacity for introduction of catalytic or similar biological functionalities. Here we describe the construction of CPs utilizing protein cage nanoparticles (PCN) as secondary building units. The dodecameric Dps protein cage from the hyperthermophilic archeon Sulfolobus solfataricus was modified for metal binding by chemical ligation of metal-chelating functionalities to the cage exterior. Treatment of modified PCN with transition metals results in the rapid formation of PCN–metal assemblies. These assemblies are characterized by a combination of dynamic light scattering, electron microscopy, small angle X-ray scattering and gas sorption studies.
[Show abstract][Hide abstract] ABSTRACT: Magnetometry was used to determine the magnetic properties of maghemite (γ -Fe <sub>2</sub> O <sub>3</sub>) nanoparticles formed within Listeria innocua protein cage. The electron magnetic resonance spectrum shows the presence of at least two magnetization components. The magnetization curves are explained by a sum of two Langevin functions in which each filled protein cage contains both a large magnetic iron oxide core plus an amorphous surface consisting of small noncoupled iron oxide spin clusters. This model qualitatively explains the observed decrease in the temperature dependent saturation moment and removes an unrealistic temperature dependent increase in the particle moment often observed in nanoparticle magnetization measurements.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14-15,000 Sanger reads per site) was obtained for five high-temperature (>65 degrees C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.
[Show abstract][Hide abstract] ABSTRACT: A branched iron-phenanthroline based coordination polymer has been constructed in a water based system using a click chemistry approach to link monomeric coordination complexes together within a protein cage nanoarchitecture, which acts both as a template and a sized constrained reaction environment.
Full-text · Article · Jan 2010 · Chemical Communications
[Show abstract][Hide abstract] ABSTRACT: Utilizing a biomimetic approach toward materials synthesis a ferritin protein cage, from the hyperthermophilic archeaon Pyrococcus furiosus, was utilized to first tempate the formation of a largely amorphous ferrihydrite iron oxide and then bring about its transformation to hematite (α-Fe2O3). This was achieved under boiling aqueous conditions by refluxing the ferritin protein cage/ferrihydrite composite. The resultant material showed diffraction indicative of hematite, a visible band gap semiconductor, and photocurrents were measured under visible illumination. The resultant protein/mineral composite was also studied via dynamic light scattering, transmission electron microscopy, and size exclusion chromatography.
Full-text · Article · Jan 2010 · Journal of Materials Chemistry