Lipopolysaccharide Density and Structure Govern the Extent and Distance of Nanoparticle Interaction with Actual and Model Bacterial Outer Membranes.

Design of nanomedicines and nanoparticle-based antimicrobial and antifouling formulations and assessment of the potential implications of nanoparticle release into the environment requires understanding nanoparticle interaction with bacterial surfaces. Here we demonstrate the electrostatically driven association of functionalized nanoparticles with lipopolysaccharides of Gram-negative bacterial outer membranes and find that lipopolysaccharide structure influences the extent and location of binding relative to the outer leaflet-solution interface. By manipulating the lipopolysaccharide content in Shewanella oneidensis outer membranes, we observed the electrostatically driven interaction of cationic gold nanoparticles with the lipopolysaccharide-containing leaflet. We probed this interaction by quartz crystal microbalance with dissipation monitoring (QCM-D) and second harmonic generation (SHG) using solid-supported lipopolysaccharide-containing bilayers. The association of cationic nanoparticles increased with lipopolysaccharide content, while no association of anionic nanoparticles was observed. The harmonic-dependence of QCM-D measurements suggested that a population of the cationic nanoparticles was held at a distance from the outer leaflet-solution interface of bilayers containing smooth lipopolysaccharides (those bearing a long O-polysaccharide). Additionally, smooth lipopolysaccharides held the bulk of the associated cationic particles outside of the interfacial zone probed by SHG. Our results demonstrate that positively charged nanoparticles are more likely to interact with Gram-negative bacteria than are negatively charged particles, and this interaction occurs primarily through lipopolysaccharides.

Attachment of pathogenic prion protein to model oxide surfaces.

Prions are the infectious agents in the class of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies, which affect humans, deer, sheep, and cattle. Prion diseases of deer and sheep can be transmitted via environmental routes, and soil is has been implicated in the transmission of these diseases. Interaction with soil particles is expected to govern the transport, bioavailability and persistence of prions in soil environments. A mechanistic understanding of prion interaction with soil components is critical for understanding the behavior of these proteins in the environment. Here, we report results of a study to investigate the interactions of prions with model oxide surfaces (Al2O3, SiO2) using quartz crystal microbalance with dissipation monitoring and optical waveguide light mode spectroscopy. The efficiency of prion attachment to Al2O3 and SiO2 depended strongly on pH and ionic strength in a manner consistent with electrostatic forces dominating interaction with these oxides. The presence of the N-terminal portion of the protein appeared to promote attachment to Al2O3 under globally electrostatically repulsive conditions. We evaluated the utility of recombinant prion protein as a surrogate for prions in attachment experiments and found that its behavior differed markedly from that of the infectious agent. Our findings suggest that prions would tend to associate with positively charged mineral surfaces in soils (e.g., iron and aluminum oxides).

Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study.

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a ß-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.

Transport of the pathogenic prion protein through soils.

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases and include bovine spongiform encephalopathy of cattle, chronic wasting disease (CWD) of deer and elk, scrapie in sheep and goats, and Creutzfeldt-Jakob disease in humans. An abnormally folded form of the prion protein (designated PrP(TSE)) is typically associated with TSE infectivity and may constitute the major, if not sole, component of the infectious agent. Transmission of CWD and scrapie is mediated in part by an environmental reservoir of infectivity. Soil appears to be a plausible candidate for this reservoir. The transport of TSE agent through soil is expected to influence the accessibility of the pathogen to animals after deposition and must be understood to assess the risks associated with burial of infected carcasses. We report the results of saturated column experiments designed to evaluate PrP(TSE) transport through five soils with relatively high sand or silt contents and low organic carbon content. Protease-treated TSE-infected brain homogenate was used as a model for PrP(TSE) present in decomposing infected tissue. Synthetic rainwater was used as the eluent. All five soils retained PrP(TSE); no detectable PrP(TSE) was eluted over more than 40 pore volumes of flow. Lower bound apparent attachment coefficients were estimated for each soil. Our results suggest that TSE agent released from decomposing tissues to soils with low organic carbon content would remain near the site of initial deposition. In the case of infected carcasses deposited on the land surface, this may result in local sources of infectivity to other animals.

Transport of the pathogenic prion protein through landfill materials.

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP(TSE)) is the major, if not sole, component of the infectious agent RecentTSE outbreaks in domesticated and wild animal populations have created the need for safe and effective disposal of large quantities of potentially infected materials. Here, we report results of a study to evaluate the potential for transport of PrP(TSE) derived from carcasses and associated wastes in municipal solid waste (MSW) landfills. Column experiments were conducted to evaluate PrP(TSE) transport in quartz sand, two fine-textured burial soils currently used in landfill practice, a green waste residual material (a potential burial material), and fresh and aged MSW. PrP(TSE) was retained by quartz sand and the fine-textured burial soils, with no detectable PrP(TSE) eluted over more than 40 pore volumes. In contrast, PrP(TSE) was more mobile in MSW and green waste residual. Transport parameters were estimated from the experimental data and used to model PrP(TSE) migration in a MSW landfill. To the extent that the PrP(TSE) used mimics that released from decomposing carcasses and the column experiments adequately simulate prion transport through burial soils, burial of CWD-infected materials at MSW landfills could provide secure containment of PrP(TSE) provided reasonable burial strategies (e.g., encasement in fine-grained soil) are used.

Gastrointestinal biodurability of engineered nanoparticles: Development of an in vitro assay.

The toxicity of engineered nanoparticles is expected to depend in part on their stability in biological systems. To assess the biodurability of engineered nanomaterials in the human digestive system, we adapted an in vitro assay previously used to evaluate the bioaccessibility of metals in contaminated soils. The compositions of the simulated gastric and intestinal fluids, temperature and residence times were designed to closely mimic conditions in the stomach and duodenum of the small intestine. We demonstrated the utility of the assay using CdSecore/ZnSshell quantum dots functionalized with polyethylene glycol (PEG) thiol of two different molecular masses (PEG350 and PEG5000). Under gastric conditions, removal of the PEG ligand diminished the stability of PEG350-quantum dot suspensions, while PEG5000-quantum dots were severely degraded. Inclusion of the glycoprotein mucin, but not the digestive protein pepsin, in simulated gastric fluids provided both PEG350- and PEG5000-coated quantum dots partial protection from transformations induced by gastric conditions.

Persistence of pathogenic prion protein during simulated wastewater treatment processes.

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a class of fatal neurodegenerative diseases affecting a variety of mammalian species including humans. A misfolded form of the prion protein (PrP(TSE)) is the major, if not sole, component of the infectious agent. Prions are highly resistant to degradation and to many disinfection procedures suggesting that, if prions enter wastewater treatment systems through sewers and/or septic systems (e.g., from slaughterhouses, necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material, prions could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and persistence of PrPTSE during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. Incubation with activated sludge did not result in significant PrPTSE degradation. PrPTSE and prion infectivity partitioned strongly to activated sludge solids and are expected to enter biosolids treatment processes. A large fraction of PrPTSE survived simulated mesophilic anaerobic sludge digestion. The small reduction in recoverable PrPTSE after 20-d anaerobic sludge digestion appeared attributable to a combination of declining extractability with time and microbial degradation. Our results suggest that if prions were to enter municipal wastewater treatment systems, most would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids.