Alzheimer's Disease: A Molecular Model and Implied Path to Improved Therapy.

Amyloid-associated neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by the in-brain accumulation of ß-sheet structured protein aggregates called amyloids. However, neither a disease model nor therapy is established. We review past data and present new, preliminary data and opinions to help solve this problem. The following is the data-derived model/hypothesis. (1) Amyloid-forming proteins have innate immunity functions implemented by conversion to another sheet conformation, α-sheet. (2) In health, α-sheet structured, amyloid-forming proteins inactivate microbes by co-assembly with microbe α-sheets. Amyloid-forming proteins then undergo α-to-ß-sheet conversion. (3) In disease, α-sheet-structured, amyloid-forming proteins over-accumulate and are neuron-toxic. This hypothesis includes formation by virus capsid subunits of α-sheets. In support, we find that 5-10 mM methylene blue (MB) at 54 °C has a hyper-expanding, thinning effect on the phage T4 capsid, as seen by negative stain- and cryo-electron microscopy after initial detection by native gel electrophoresis (AGE). Given the reported mild anti-AD effect of MB, we propose the following corollary hypothesis. (1) Anti-AD MB activity is, at least in part, caused by MB-binding to amyloid α-sheet and (2) MB induces the transition to α-sheet of T4 capsid subunits. We propose using AGE of drug incubated T4 to test for improved anti-AD activity.

Extracellular Interaction of Bacillus thuringiensis, ATP and Phage 0105phi7-2: A Potential New Anti-Bacterial Strategy.

The following hypothesis proposes non-diffusive, environmental bacteriophage (phage) motion. (1) Some phage-hosting, motile bacteria undergo chemotaxis down ATP concentration gradients to escape lysis-inducing conditions, such as phage infection. (2) Some phages respond by non-infective binding to the motile bacteria. (3) When the bacteria reach a lower ATP concentration, which is a condition that signals increased density of phage-susceptible bacteria, the phage converts, Trojan-horse-like, to productive binding and infection. This hypothesis was previously proposed for Bacillus thuringiensis siphophage 0105phi7-2. It is tested here and confirmed with the following observations. (1) B. thuringiensis is found, macroscopically, preferentially located at low ATP concentrations when propagated in-gel after inoculation in the center of an artificially generated ATP concentration gradient. (2) Inoculating phage 0105phi7-2 at the bacteria inoculation site, 2-3 h after inoculation of bacteria, results in cell lysing activity that moves with the bacteria, without a visible trail of lysis. Trojan-horse-like behavior is consistent with only biofilm-inhabiting phages because environmental selection for this behavior requires limited fluid flows. We propose using artificial ATP concentration gradients to instigate Trojan-horse-like phage behavior during phage therapy of bacterial biofilms.

Siphophage 0105phi7-2 of Bacillus thuringiensis: Novel Propagation, DNA, and Genome-Implied Assembly.

Diversity of phage propagation, physical properties, and assembly promotes the use of phages in ecological studies and biomedicine. However, observed phage diversity is incomplete. Bacillus thuringiensis siphophage, 0105phi-7-2, first described here, significantly expands known phage diversity, as seen via in-plaque propagation, electron microscopy, whole genome sequencing/annotation, protein mass spectrometry, and native gel electrophoresis (AGE). Average plaque diameter vs. plaque-supporting agarose gel concentration plots reveal unusually steep conversion to large plaques as agarose concentration decreases below 0.2%. These large plaques sometimes have small satellites and are made larger by orthovanadate, an ATPase inhibitor. Phage head-host-cell binding is observed by electron microscopy. We hypothesize that this binding causes plaque size-increase via biofilm evolved, ATP stimulated ride-hitching on motile host cells by temporarily inactive phages. Phage 0105phi7-2 does not propagate in liquid culture. Genomic sequencing/annotation reveals history as temperate phage and distant similarity, in a virion-assembly gene cluster, to prototypical siphophage SPP1 of Bacillus subtilis. Phage 0105phi7-2 is distinct in (1) absence of head-assembly scaffolding via either separate protein or classically sized, head protein-embedded peptide, (2) producing partially condensed, head-expelled DNA, and (3) having a surface relatively poor in AGE-detected net negative charges, which is possibly correlated with observed low murine blood persistence.

A Perspective on Studies of Phage DNA Packaging Dynamics.

The Special Issue "DNA Packaging Dynamics of Bacteriophages" is focused on an event that is among the physically simplest known events with biological character. Thus, phage DNA (and RNA) packaging is used as a relatively accessible model for physical analysis of all biological events. A similar perspective motivated early phage-directed work, which was a major contributor to early molecular biology. However, analysis of DNA packaging encounters the limitation that phages vary in difficulty of observing various aspects of their packaging. If a difficult-to-access aspect arises while using a well-studied phage, a counterstrategy is to (1) look for and use phages that provide a better access "window" and (2) integrate multi-phage-accessed information with the help of chemistry and physics. The assumption is that all phages are characterized by the same evolution-derived themes, although with variations. Universal principles will emerge from the themes. A spin-off of using this strategy is the isolation and characterization of the diverse phages needed for biomedicine. Below, I give examples in the areas of infectious disease, cancer, and neurodegenerative disease.

Additions to Alpha-Sheet Based Hypotheses for the Cause of Alzheimer's Disease.

Protein amyloid-ß (Aß) oligomers with ß-sheet-like backbone (ß-structured) form extracellular amyloid plaques associated with Alzheimer's disease (AD). However, the relationship to AD is not known. Some investigations suggest that the toxic Aß component has α-sheet-like backbone (α-structured) subsequently detoxified by intracellular α-to-ß conversion before plaque formation. Our objective is to compare this latter hypothesis with observations made by electron microscopy of thin sections of AD-cerebral cortex. We observe irregular, 200-2,000 nm, intracellular, lipofuscin-like inclusions. Some are light-staining and smooth. Others are dark-staining and made granular by fibers that are usually overlapping and are sometimes individually seen. Aspects unusual for lipofuscin include 1) dark and light inclusions interlocking as though previously one inclusion, 2) dark inclusion-contained 2.6 nm thick sub-fibers that are bent as though α-structured, and 3) presence of inclusions in lysosomes and apparent transfer of dark inclusion material to damaged, nearby lysosomal membranes. These data suggest the following additions to α-structure-based hypotheses: 1) Lipofuscin-associated, α-structured protein toxicity to lysosomal membranes is in the chain of AD causation; 2) α-to-ß detoxification of α-structured protein occurs in lipofuscin and causes dark-to-light transition that, when incomplete, is the origin of cell-to-cell transmission essential for development of AD.

Structural Studies of the Phage G Tail Demonstrate an Atypical Tail Contraction.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host-phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head-neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head-neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G's sheath structure in the non-contracted and contracted states are similar to phage T4's sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath's helical symmetry, the sheath protein's evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head-neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Basics for Improved Use of Phages for Therapy.

Blood-borne therapeutic phages and phage capsids increasingly reach therapeutic targets as they acquire more persistence, i.e., become more resistant to non-targeted removal from blood. Pathogenic bacteria are targets during classical phage therapy. Metastatic tumors are potential future targets, during use of drug delivery vehicles (DDVs) that are phage derived. Phage therapy has, to date, only sometimes been successful. One cause of failure is low phage persistence. A three-step strategy for increasing persistence is to increase (1) the speed of lytic phage isolation, (2) the diversity of phages isolated, and (3) the effectiveness and speed of screening phages for high persistence. The importance of high persistence-screening is illustrated by our finding here of persistence dramatically higher for coliphage T3 than for its relative, coliphage T7, in murine blood. Coliphage T4 is more persistent, long-term than T3. Pseudomonas chlororaphis phage 201phi2-1 has relatively low persistence. These data are obtained with phages co-inoculated and separately assayed. In addition, highly persistent phage T3 undergoes dispersal to several murine organs and displays tumor tropism in epithelial tissue (xenografted human oral squamous cell carcinoma). Dispersal is an asset for phage therapy, but a liability for phage-based DDVs. We propose increased focus on phage persistence-and dispersal-screening.

Simulations of Phage T7 Capsid Expansion Reveal the Role of Molecular Sterics on Dynamics.

Molecular dynamics techniques provide numerous strategies for investigating biomolecular energetics, though quantitative analysis is often only accessible for relatively small (frequently monomeric) systems. To address this limit, we use simulations in combination with a simplified energetic model to study complex rearrangements in a large assembly. We use cryo-EM reconstructions to simulate the DNA packaging-associated 3 nm expansion of the protein shell of an initially assembled phage T7 capsid (called procapsid or capsid I). This is accompanied by a disorder-order transition and expansion-associated externalization displacement of the 420 N-terminal tails of the shell proteins. For the simulations, we use an all-atom structure-based model (1.07 million atoms), which is specifically designed to probe the influence of molecular sterics on dynamics. We find that the rate at which the N-terminal tails undergo translocation depends heavily on their position within hexons and pentons. Specifically, trans-shell displacements of the hexon E subunits are the most frequent and hexon A subunits are the least frequent. The simulations also implicate numerous tail translocation intermediates during tail translocation that involve topological traps, as well as sterically induced barriers. The presented study establishes a foundation for understanding the precise relationship between molecular structure and phage maturation.

Using the Past to Maximize the Success Probability of Future Anti-Viral Vaccines.

Rapid obtaining of safe, effective, anti-viral vaccines has recently risen to the top of the international agenda. To maximize the success probability of future anti-viral vaccines, the anti-viral vaccines successful in the past are summarized here by virus type and vaccine type. The primary focus is on viruses with both single-stranded RNA genomes and a membrane envelope, given the pandemic past of influenza viruses and coronaviruses. The following conclusion is reached, assuming that success of future strategies is positively correlated with strategies successful in the past. The primary strategy, especially for emerging pandemic viruses, should be development of vaccine antigens that are live-attenuated viruses; the secondary strategy should be development of vaccine antigens that are inactivated virus particles. Support for this conclusion comes from the complexity of immune systems. These conclusions imply the need for a revision in current strategic planning.

Optimizing Anti-Viral Vaccine Responses: Input from a Non-Specialist.

Recently, the research community has had a real-world look at reasons for improving vaccine responses to emerging RNA viruses. Here, a vaccine non-specialist suggests how this might be done. I propose two alternative options and compare the primary alternative option with current practice. The basis of comparison is feasibility in achieving what we need: a safe, mass-produced, emerging virus-targeted vaccine on 2-4 week notice. The primary option is the following. (1) Start with a platform based on live viruses that infect bacteria, but not humans (bacteriophages, or phages). (2) Isolate phages (to be called pathogen homologs) that resemble and provide antigenic context for membrane-covered, pathogenic RNA viruses; coronavirus-phage homologs will probably be found if the search is correctly done. (3) Upon isolating a viral pathogen, evolve its phage homolog to bind antibodies neutralizing for the viral pathogen. Vaccinate with the evolved phage homolog by generating a local, non-hazardous infection with the phage host and then curing the infection by propagating the phage in the artificially infecting bacterial host. I discuss how this alternative option has the potential to provide what is needed after appropriate platforms are built.

Phage G Structure at 6.1 ŠResolution, Condensed DNA, and Host Identity Revision to a Lysinibacillus.

Phage G has the largest capsid and genome of any known propagated phage. Many aspects of its structure, assembly, and replication have not been elucidated. Herein, we present the dsDNA-packed and empty phage G capsid at 6.1 and 9 Šresolution, respectively, using cryo-EM for structure determination and mass spectrometry for protein identification. The major capsid protein, gp27, is identified and found to share the HK97-fold universally conserved in all previously solved dsDNA phages. Trimers of the decoration protein, gp26, sit on the 3-fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G's decoration protein is longer than what has been reported in other phages, and we suspect the extra interaction surface area helps stabilize the capsid. We identified several additional capsid proteins, including a candidate for the prohead protease responsible for processing gp27. Furthermore, cryo-EM reveals a range of partially full, condensed DNA densities that appear to have no contact with capsid shell. Three analyses confirm that the phage G host is a Lysinibacillus, and not Bacillus megaterium: identity of host proteins in our mass spectrometry analyses, genome sequence of the phage G host, and host range of phage G.

In-Gel Isolation and Characterization of Large (and Other) Phages.

We review some aspects of the rapid isolation of, screening for and characterization of jumbo phages, i.e., phages that have dsDNA genomes longer than 200 Kb. The first aspect is that, as plaque-supporting gels become more concentrated, jumbo phage plaques become smaller. Dilute agarose gels are better than conventional agar gels for supporting plaques of both jumbo phages and, prospectively, the even larger (>520 Kb genome), not-yet-isolated mega-phages. Second, dilute agarose gels stimulate propagation of at least some jumbo phages. Third, in-plaque techniques exist for screening for both phage aggregation and high-in-magnitude, negative average electrical surface charge density. The latter is possibly correlated with high phage persistence in blood. Fourth, electron microscopy of a thin section of a phage plaque reveals phage type, size and some phage life cycle information. Fifth, in-gel propagation is an effective preparative technique for at least some jumbo phages. Sixth, centrifugation through sucrose density gradients is a relatively non-destructive jumbo phage purification technique. These basics have ramifications in the development of procedures for (1) use of jumbo phages for phage therapy of infectious disease, (2) exploration of genomic diversity and evolution and (3) obtaining accurate metagenomic analyses.

Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers.

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

High murine blood persistence of phage T3 and suggested strategy for phage therapy.

OBJECTIVE: Our immediate objective is to determine whether infectivity of lytic podophage T3 has a relatively high persistence in the blood of a mouse, as suggested by previous data. Secondarily, we determine whether the T3 surface has changed during this mouse passage. The surface is characterized by native agarose gel electrophoresis (AGE). Beyond our current data, the long-term objective is optimization of phages chosen for therapy of all bacteremias and associated sepsis. RESULTS: We find that the persistence of T3 in mouse blood is higher by over an order of magnitude than the previously reported persistence of (1) lysogenic phages lambda and P22, and (2) lytic phage T7, a T3 relative. We explain these differences via the lysogenic character of lambda and P22, and the physical properties of T7. For the future, we propose testing a new, AGE-based strategy for rapidly screening for high-persistence, lytic, environmental podophages that have phage therapy-promoting physical properties.

Cell-gel interactions of in-gel propagating bacteria.

OBJECTIVE: Our immediate objective is to test the data-suggested possibility that in-agarose gel bacterial propagation causes gel fiber dislocation and alteration of cell distribution. We also test the further effect of lowering water activity. We perform these tests with both Gram-negative and Gram-positive bacteria. Data are obtained via electron microscopy of thin sections, which provides the first images of both bacteria and gel fibers in gel-supported bacterial lawns. The long-term objective is analysis of the effects of in-gel propagation on the DNA packaging of phages. RESULTS: We find that agarose gel-supported cells in lawns of Escherichia coli and Lysinibacillus (1) are primarily in clusters that increase in size with time and are surrounded by gel fibers, and (2) sometimes undergo gel-induced, post-duplication rotation and translation. Bacterial growth-induced dislocation of gel fibers is observed. One reason for clustering is that clustering promotes growth by increasing the growth-derived force applied to the gel fibers. Reactive force exerted by gel on cells explains cell movement. Finally, addition to growth medium of 0.94 M sucrose causes cluster-associated E. coli cells to become more densely packed and polymorphic. Shape is determined, in part, by neighboring cells, a novel observation to our knowledge.

Nanomedicine and Phage Capsids.

Studies of phage capsids have at least three potential interfaces with nanomedicine. First, investigation of phage capsid states potentially will provide therapies targeted to similar states of pathogenic viruses. Recently detected, altered radius-states of phage T3 capsids include those probably related to intermediate states of DNA injection and DNA packaging (dynamic states). We discuss and test the idea that some T3 dynamic states include extensive α-sheet in subunits of the capsid’s shell. Second, dynamic states of pathogenic viral capsids are possible targets of innate immune systems. Specifically, α-sheet-rich innate immune proteins would interfere with dynamic viral states via inter-α-sheet co-assembly. A possible cause of neurodegenerative diseases is excessive activity of these innate immune proteins. Third, some phage capsids appear to have characteristics useful for improved drug delivery vehicles (DDVs). These characteristics include stability, uniformity and a gate-like sub-structure. Gating by DDVs is needed for (1) drug-loading only with gate opened; (2) closed gate-DDV migration through circulatory systems (no drug leakage-generated toxicity); and (3) drug release only at targets. A gate-like sub-structure is the connector ring of double-stranded DNA phage capsids. Targeting to tumors of phage capsid-DDVs can possibly be achieved via the enhanced permeability and retention effect.

Hypothesis for the cause and therapy of neurodegenerative diseases.

The cause and therapy of neurodegenerative diseases remain unsolved puzzles. These diseases are correlated with presence of beta sheet-rich amyloid assemblies. Here, I derive and assemble puzzle pieces to obtain a loose end-tying hypothesis for cause with direct implications for therapy. I use the following extrapolations to find connectable puzzle pieces: (a) the traditional extrapolation that amyloid/amyloid precursors cause disease, (b) a recent extrapolation that amyloid-forming proteins, some of which are virus protein homologs, are components of an empirically obscure innate immune system that counters insults, including those by both viruses and bacteria, (c) a new extrapolation that various insults produce assemblies with structural features in common and that amyloid-forming, innate immune system proteins recognize these features and, then, counter insults by co-assembly, (d, 1) a second new extrapolation that beta sheet is a common structural feature and is extended during insult-neutralizing co-assembly and (d, 2) an appendix, derived from studies of phages T3 and T4, that most insult-produced assemblies are obscure to current biochemical analysis. The hypothesis is the following. One function of amyloid-forming proteins is non-classical innate immunity to biological insults. This immunity works via beta sheet-extending co-assembly of amyloid-forming proteins with beta sheet-containing insult products. For example, co-assembly with beta sheet-containing viral assembly intermediates inhibits virus production. Amyloid-forming proteins cause neurodegenerative disease when errant, typically overproduced. Other innate immunity systems sometimes exacerbate symptoms. This hypothesis suggests the following therapy, based on manipulating Nature's chemistry. First, conduct directed evolution to obtain low-pathogenicity, chronic symptom-producing viruses with assembly intermediates that co-assemble with and destabilize both amyloid and amyloid sub-assemblies. Then, infect patients with these viruses.

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM.

Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo.

Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Restoring logic and data to phage-cures for infectious disease.

Antibiotic therapy for infectious disease is being compromised by emergence of multi-drug-resistant bacterial strains, often called superbugs. A response is to use a cocktail of several bacteria-infecting viruses (bacteriophages or phages) to supplement antibiotic therapy. Use of such cocktails is called phage therapy, which has the advantage of response to bacterial resistance that is rapid and not exhaustible. A procedure of well-established success is to make cocktails from stockpiles of stored environmental phages. New phages are added to stockpiles when phage therapy becomes thwarted. The scientific subtext includes optimizing the following aspects: (1) procedure for rapidly detecting, purifying, storing and characterizing phages for optimization of phage cocktails, (2) use of directed evolution in the presence of bacteriostatic compounds to obtain phages that can be most efficiently used for therapy in the presence of these compounds, (3) phage genome sequencing technology and informatics to improve the characterization of phages, and (4) database technology to make optimal use of all relevant information and to rapidly retrieve phages for cocktails that will vary with the infection(s) involved. The use of phage stockpiles has an established record, including a recent major human-therapy success by the US Navy. However, I conclude that most research is not along this track and, therefore, is not likely to lead to real world success. I find that a strong case exists for action to rectify this situation.