Janus-like protein cages. Spatially controlled dual-functional surface modifications of protein cages.

Protein cages have been used both as size-constrained reaction vessels for nanomaterials synthesis and as nanoscale building blocks for higher order nanostructures. We generated Janus-like protein cages, which are dual functionalized with a fluorescent and an affinity label, and demonstrated control over both the stoichiometry and spatial distribution of the functional groups. The capability to toposelectively functionalize protein cages has allowed us to manipulate hierarchical assembly using the layer-by-layer assembly process. Janus-like protein cages expand the toolkit of nanoplatforms that can be used for directed assembly of nanostructured materials.

A streptavidin-protein cage Janus particle for polarized targeting and modular functionalization.

The incorporation of Janus particles into the repertoire of nanoscale building blocks adds a new level of control to supramolecular assembly. Here we demonstrate the potential for using toposelective modification to assemble new types of targeting nanoplatforms by docking the universal coupling protein, streptavidin (StAv), onto a restricted region of the surface of a small protein cage. The resulting StAv-functionalized Janus particles have the potential to be used to control the orientation of the nanoplatforms targeted to a cell surface. In addition, the StAv-biotin couple provides an ideal molecular adaptor for extending asymmetric (polarized) supramolecular assembly. To demonstrate this potential application, StAv-functionalized nanoplatforms were coupled to a biotinylated monoclonal antibody and used to target a microbial pathogen.

Modular spectral imaging system for discrimination of pigments in cells and microbial communities.

Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.

A Candida albicans early stage biofilm detachment event in rich medium.

BACKGROUND: Dispersal from Candida albicans biofilms that colonize catheters is implicated as a primary factor in the link between contaminated catheters and life threatening blood stream infections (BSI). Appropriate in vitro C. albicans biofilm models are needed to probe factors that induce detachment events. RESULTS: Using a flow through system to culture C. albicans biofilms we characterized a detachment process which culminates in dissociation of an entire early stage biofilm from a silicone elastomer surface. We analyzed the transcriptome response at time points that bracketed an abrupt transition in which a strong adhesive association with the surface is weakened in the initial stages of the process, and also compared batch and biofilm cultures at relevant time points. K means analysis of the time course array data revealed categories of genes with similar patterns of expression that were associated with adhesion, biofilm formation and glycoprotein biosynthesis. Compared to batch cultures the biofilm showed a pattern of expression of metabolic genes that was similar to the C. albicans response to hypoxia. However, the loss of strong adhesion was not obviously influenced by either the availability of oxygen in the medium or at the silicone elastomer surface. The detachment phenotype of mutant strains in which selected genes were either deleted or overexpressed was characterized. The microarray data indicated that changes associated with the detachment process were complex and, consistent with this assessment, we were unable to demonstrate that transcriptional regulation of any single gene was essential for loss of the strong adhesive association. CONCLUSION: The massive dispersal of the early stage biofilm from a biomaterial surface that we observed is not orchestrated at the level of transcriptional regulation in an obvious manner, or is only regulated at this level by a small subpopulation of cells that mediate adhesion to the surface.

Photosensitizer efficiency in genetically modified protein cage architectures.

A Ru(bpy)(3)(2+) photosensitizer was covalently linked at specific sites on the interior or exterior surface of genetic constructs of a small heat shock protein cage nanoplatform and the light activated production of singlet oxygen was characterized.

High-density targeting of a viral multifunctional nanoplatform to a pathogenic, biofilm-forming bacterium.

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.

Candida albicans viability after exposure to amphotericin B: assessment using metabolic assays and colony forming units.

Metabolic assays are a preferred method for evaluation of Candida albicans viability after exposure to antimicrobial agents in cases in which the culture is a complex mixture of yeast and filamentous forms. There is a lack of published data indicating the strength of the correlation between metabolic assays and viable cell numbers determined by a standard assay such as colony forming units (CFU). We developed a kinetic metabolic assay (KMA) for quantifying viable cells which was tested on yeast cells in both exponential and stationary phase using alamarBlue and XTT as metabolic indicators. The KMA enabled quantification of the viable population over a range of 10(1) to 10(7) cells that linearly correlated (R(2)>0.98) with estimates made by enumeration of CFU regardless of the indicator or growth phase of the cells. Linear relationships were used to calibrate the KMA in terms of equivalent CFU. Viable cell populations were then determined after exposure to AmB. These results were compared with those obtained by direct enumeration of CFU. There were significant correlations between KMA-derived equivalent CFU and direct CFU estimates of viable cell populations for exponential-phase cells. However, the proportions of viable cells based on the KMA were consistently lower than those obtained directly by CFU. This trend was substantially more pronounced for stationary phase cells. These results show that even in the relatively simple case in which only the yeast form is present, the relationship between assessment by metabolic assays and CFU is perturbed by exposure to an antimicrobial and that, furthermore, growth phase alters the nature of the perturbation.

A method for discrimination of subpopulations of Candida albicans biofilm cells that exhibit relative levels of phenotypic resistance to chlorhexidine.

Microbes in biofilms are generally found to be resistant to antimicrobial agents. One set of hypotheses attributes biofilm resistance to acquisition of special physiological traits (phenotypic resistance). Methods are presented that allow discrimination of subpopulations of Candida albicans cells that exhibit relative levels of phenotypic resistance to chlorhexidine. The assay for phenotypic resistance is based on microscopic detection of the rate of penetration of propidium iodide (PI) into single cells as their membranes become disrupted by chlorhexidine. Using the assay, it was found that batch cultures became progressively more resistant to the action of chlorhexidine during the transition from exponential growth to early stationary phase. Results are presented demonstrating that the methods can be used to characterize relative levels of phenotypic resistance exhibited by cells at the base of a C. albicans biofilm.

A review of spectroscopic methods for characterizing microbial transformations of minerals.

Over the past decade, advances in surface-sensitive spectroscopic techniques have provided the opportunity to identify many new microbiologically mediated biogeochemical processes. Although a number of surface spectroscopic techniques require samples to be dehydrated, which precludes real-time measurement of biotransformations and generate solid phase artifacts, some now offer the opportunity to either isolate a hydrated sample within an ultrahigh vacuum during analysis or utilize sources of radiation that efficiently penetrate hydrated specimens. Other nondestructive surface spectroscopic techniques permit determination of the influence of microbiological processes on the kinetics and thermodynamics of geochemical reactions. The ability to perform surface chemical analyses at micrometer and nanometer scales has led to the realization that bacterial cell surfaces are active sites of mineral nucleation and propagation, resulting in the formation of both stable and transient small-scale surface chemical heterogeneities. Some surface spectroscopic instrumentation is now being modified for use in the field to permit researchers to evaluate mineral biotransformations under in situ conditions. Surface spectroscopic techniques are thus offering a variety of opportunities to yield new information on the way in which microorganisms have influenced geochemical processes on Earth over the last 4 billion years.

Correct charge state assignment of native electrospray spectra of protein complexes.

Correct charge state assignment is crucial to assigning an accurate mass to supramolecular complexes analyzed by electrospray mass spectrometry. Conventional charge state assignment techniques fall short of reliably and unambiguously predicting the correct charge state for many supramolecular complexes. We provide an explanation of the shortcomings of the conventional techniques and have developed a robust charge state assignment method that is applicable to all spectra.

Targeting and photodynamic killing of a microbial pathogen using protein cage architectures functionalized with a photosensitizer.

The selectivity of antimicrobial photodynamic therapy (PDT) can be enhanced by coupling the photosensitizer (PS) to a targeting ligand. Nanoplatforms provide a medium for designing delivery vehicles that incorporate both functional attributes. We report here the photodynamic inactivation of a pathogenic bacterium, Staphylococcus aureus, using targeted nanoplatforms conjugated to a photosensitizer (PS). Both electrostatic and complementary biological interactions were used to mediate targeting. Genetic constructs of a protein cage architecture allowed site-specific chemical functionalization with the PS and facilitated dual functionalization with the PS and the targeting ligand. These results demonstrate that protein cage architectures can serve as versatile templates for engineering nanoplatforms for targeted antimicrobial PDT.

Assembly of multilayer films incorporating a viral protein cage architecture.

Protein cage architectures such as viral capsids, heat shock proteins, ferritins, and DNA-binding proteins are nanoscale modular subunits that can be used to expand the structural and functional range of composite materials. Here, layer-by-layer (LbL) assembly was used to incorporate cowpea chlorotic mottle virus (CCMV) into multilayer films. Three types of multilayer films were prepared. In the first type, ionic interactions were employed to assemble CCMV into triple layers. In the second type, complementary biological interactions (streptavidin/biotin) were used for this purpose. In a third variation of LbL assembly, complementary biological interactions were employed to produce nanotextured films that exhibit in-plane order over a micron scale without the need to adsorb onto a prepatterned template.

A small subpopulation of blastospores in candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes.

The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential- or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and beta-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.

Influence of electrostatic interactions on the surface adsorption of a viral protein cage.

The Cowpea chlorotic mottle virus (CCMV) provides a useful protein-cage architecture that can be used for the size- and shape-constrained chemistry of nanomaterials. The control of surface assembly is necessary for the realization of many applications of these nanoscale reaction vessels. Electrostatic interactions provide a useful (and reversible) method for controlled surface assembly. CCMV absorption behavior was studied on Formvar, bare Si, Formvar-coated Si, and Si modified by aminopropyltriethoxysilane (APS). Transmission electron microscopy (TEM), atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) were used to characterize the CCMV surface adsorption. Combined AFM and ATR-FTIR data indicated that the viral coverage on the modified surfaces was approximately 84% of the jamming limit predicted by the random sequential adsorption (RSA) model. According to the ATR-FTIR results, surface coverage was not increased at higher ionic strengths nor at a pH near the isoelectric point (pI) of the virus. The Langmuir model was used to provide a description of the kinetic absorption behavior.

Action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage Candida albicans biofilm.

An in situ method for sensitive detection of differences in the action of chlorhexidine against subpopulations of cells in Candida albicans biofilms is described. Detection relies on monitoring the kinetics of propidium iodide (PI) penetration into the cytoplasm of individual cells during dosing with chlorhexidine. Accurate estimation of the time for delivery of the dosing concentration to the substratum was facilitated by using a flow cell system for which transport to the interfacial region was previously characterized. A model was developed to quantify rates of PI penetration based on the shape of the kinetic data curves. Yeast were seeded onto the substratum, and biofilm formation was monitored microscopically for 3 h. During this period a portion of the yeast germinated, producing filamentous forms (both hyphae and pseudohyphae). When the population was subdivided on the basis of cell morphology, rates of PI penetration into filamentous forms appeared to be substantially higher than for yeast forms. Based on the model, rates of penetration were assigned to individual cells. These data indicated that the difference in rates between the two subpopulations was statistically significant (unpaired t test, P < 0.0001). A histogram of rates and analysis of variance indicated that rates were approximately equally distributed among different filamentous forms and between apical and subapical segments of filamentous forms.