The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin.

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium-phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey-predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.

Antiparallel triple-strand architecture for prefibrillar Aß42 oligomers.

Aß42 oligomers play key roles in the pathogenesis of Alzheimer disease, but their structures remain elusive partly due to their transient nature. Here, we show that Aß42 in a fusion construct can be trapped in a stable oligomer state, which recapitulates characteristics of prefibrillar Aß42 oligomers and enables us to establish their detailed structures. Site-directed spin labeling and electron paramagnetic resonance studies provide structural restraints in terms of side chain mobility and intermolecular distances at all 42 residue positions. Using these restraints and other biophysical data, we present a novel atomic-level oligomer model. In our model, each Aß42 protein forms a single ß-sheet with three ß-strands in an antiparallel arrangement. Each ß-sheet consists of four Aß42 molecules in a head-to-tail arrangement. Four ß-sheets are packed together in a face-to-back fashion. The stacking of identical segments between different ß-sheets within an oligomer suggests that prefibrillar oligomers may interconvert with fibrils via strand rotation, wherein ß-strands undergo an ∼90° rotation along the strand direction. This work provides insights into rational design of therapeutics targeting the process of interconversion between toxic oligomers and non-toxic fibrils.

Prion domain of yeast Ure2 protein adopts a completely disordered structure: a solid-support EPR study.

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer's, Parkinson's, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer's amyloid-ß monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.

Quantitative analysis of spin exchange interactions to identify ß strand and turn regions in Ure2 prion domain fibrils with site-directed spin labeling.

Amyloid formation is associated with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register ß structure. With quantitative analysis of spin exchange interactions, we show that residues 8-12 form the first ß strand, followed by a turn at residues 13-14, and then the second ß strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of ß-strand and turn regions in this work suggests that quantitative analysis of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the ß-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.

Solid-support electron paramagnetic resonance (EPR) studies of Aß40 monomers reveal a structured state with three ordered segments.

Alzheimer disease is associated with the pathological accumulation of amyloid-ß peptide (Aß) in the brain. Soluble Aß oligomers formed during early aggregation process are believed to be neurotoxins and causative agents in Alzheimer disease. Aß monomer is the building block for amyloid assemblies. A comprehensive understanding of the structural features of Aß monomer is crucial for delineating the mechanism of Aß oligomerization. Here we investigated the structures of Aß40 monomer using a solid-support approach, in which Aß40 monomers are tethered on the solid support via an N-terminal His tag to prevent further aggregation. EPR spectra of tethered Aß40 with spin labels at 18 different positions show that Aß40 monomers adopt a completely disordered structure under denaturing conditions. Under native conditions, however, EPR spectra suggest that Aß40 monomers adopt both a disordered state and a structured state. The structured state of Aß40 monomer has three more ordered segments at 14-18, 29-30, and 38-40. Interactions between these segments may stabilize the structured state, which likely plays an important role in Aß aggregation.

Key residues for the oligomerization of Aß42 protein in Alzheimer's disease.

Deposition of amyloid fibrils consisting of amyloid ß (Aß) protein as senile plaques in the brain is a pathological hallmark of Alzheimer's disease. However, a growing body of evidence shows that soluble Aß oligomers correlate better with dementia than fibrils, suggesting that Aß oligomers may be the primary toxic species. The structure and oligomerization mechanism of these Aß oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of Aß42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of Aß sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8M urea at pH 10, is unfavorable for Aß42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that Aß42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS-PAGE shows that Aß42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, Aß40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of Aß42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for Aß42 oligomerization.

Hierarchical organization in the amyloid core of yeast prion protein Ure2.

Formation of amyloid fibrils is involved in a range of fatal human disorders including Alzheimer, Parkinson, and prion diseases. Yeast prions, despite differences in sequence from their mammalian counterparts, share similar features with mammalian prions including infectivity, prion strain phenomenon, and species barrier and thus are good model systems for human prion diseases. Yeast prions normally have long prion domains that presumably form multiple ß strands in the fibril, and structural knowledge about the yeast prion fibrils has been limited. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structures of amyloid fibrils of Ure2 prion domain. We show that 15 spin-labeled Ure2 mutants, with spin labels at every 5th residue from position 5 to position 75, show a single-line or nearly single-line feature in their EPR spectra as a result of strong spin exchange interactions. These results suggest that a parallel in-register ß structure exists at these spin-labeled positions. More interestingly, we also show that residues in the segment 30-65 have stronger spin exchange interactions, higher local stability, and lower solvent accessibility than segments 5-25 and 70-75, suggesting different local environment at these segments. We propose a hierarchical organization in the amyloid core of Ure2, with the segment 30-65 forming an inner core and the segments 5-25 and 70-75 forming an outer core. The hierarchical organization in the amyloid core may be a structural origin for polymorphism in fibrils and prion strains.