α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

The bacterial flagellar filament has long been studied to understand how a polymer composed of a single protein can switch between different supercoiled states with high cooperativity. Here we present near-atomic resolution cryo-EM structures for flagellar filaments from both Gram-positive Bacillus subtilis and Gram-negative Pseudomonas aeruginosa. Seven mutant flagellar filaments in B. subtilis and two in P. aeruginosa capture two different states of the filament. These reliable atomic models of both states reveal conserved molecular interactions in the interior of the filament among B. subtilis, P. aeruginosa and Salmonella enterica. Using the detailed information about the molecular interactions in two filament states, we successfully predict point mutations that shift the equilibrium between those two states. Further, we observe the dimerization of P. aeruginosa outer domains without any perturbation of the conserved interior of the filament. Our results give new insights into how the flagellin sequence has been “tuned” over evolution.

Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (CDV), is being developed as a treatment for smallpox. In the absence of human cases of smallpox, new treatments must be tested for efficacy in animal models. Previously, we demonstrated the efficacy of CMX001 in protecting New Zealand White rabbits from mortality following intradermal infection with rabbitpox virus as a model for smallpox, monkeypox and for treatment of adverse reactions to smallpox vaccination. Here we extend these studies by exploring different dosing regimens and performing randomized, blinded, placebo-controlled studies. In addition, because rabbitpox virus can be transmitted via naturally generated aerosols (animal to animal transmission), we report on studies to test the efficacy of CMX001 in protecting rabbits from lethal rabbitpox virus disease when infection occurs by animal to animal transmission. In all cases, CMX001 treatment was initiated at the onset of observable lesions in the ears to model the use of CMX001 as a treatment for symptomatic smallpox. The results demonstrate that CMX001 is an effective treatment for symptomatic rabbitpox virus infection. The rabbitpox model has key similarities to human smallpox including an incubation period, generalized systemic disease, the occurrence of lesions which may be used as a trigger for initiating therapy, and natural animal to animal spread, making it an appropriate model.

Summary Imaging host-pathogen interactions in real time can provide significant insight into dynamic processes and provide information about time and space of their occurences. Here we present detailed experimental instructions on how to image the membrane penetration process of the non-enveloped adenovirus in rel time. The system is based on a cell line stably expressing the lectin galectin-3 fused to a fluorophore. Membrane-lytic events during adenovirus cell entry can be monitored by the recruitment of galectin-3 to galactose-containing membrane glycoproteins on the exo-surface of ruptured membranes. The simultaneous use of fluorescently labeled adenoviral capsids allows to image the events in unmatched temporal resolution.

The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. IMPORTANCEThese results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents.

Bacterial flagella contain an axle-like rod that transits the cell envelope and connects the transmembrane basal body to the extracellular hook and filament. Although the rod is a crucial component of the flagellum, its structure and assembly are poorly understood. Previous reports defining the order of rod assembly in Gram-negative bacteria suggest that the rod requires five proteins to successfully assemble but assembly intermediates have not been well characterized due to metastablity and periplasmic proteolysis. is a Gram-positive, genetically tractable model bacterium that synthesizes flagella and lacks a true periplasm. Here we genetically, biochemically, and cytologically determine the assembly order of the flagellar rod from cell proximal to distal as FliE, FlgB, FlgC, FlhO, and FlhP. We further show under conditions in which rod structure cannot be completed, assembly intermediates are both metastable and subject to proteolysis. Finally, we support previous results that FliE serves as both a structural assembly platform for the rod and as an enhancer of flagellar type III secretion. Bacteria rotate propeller like flagella to find and colonize environmental niches. The flagellum is a complex machine and understanding of its structure is still incomplete. Here we characterize and biochemically define the assembly order of the subunits that make up the axle-like rod. The rod is a critical component for the assembly of subsequent components and is central to our understanding of how the flagellum is anchored but still free spinning within the context of the cell envelope.

Vaccinia virus deleted for the innate immune evasion gene, E3L, has been shown to be highly attenuated and yet induces a protective immune response against challenge by homologous virus in a mouse model. In this manuscript the NYCBH vaccinia virus vaccine strain was compared to NYCBH vaccinia virus deleted for E3L (NYCBHΔE3L) in a rabbitpox virus (RPV) challenge model. Upon scarification, both vaccines produced a desired skin lesion, although the lesion produced by NYCBHΔE3L was smaller. Both vaccines fully protected rabbits against lethal challenge by escalating doses of RPV, from 10 LD50 to 1,000 LD50. A single dose of NYCBHΔE3L protected rabbits from weight loss, fever, and clinical symptoms following the lowest dose challenge of 10 LD50, however it allowed a moderate level of RPV replication at the challenge site, some spread to external skin and mucosal surfaces, and increased numbers of secondary lesions as compared to vaccination with NYCBH. Alternately, two doses of NYCBHΔE3L fully protected rabbits from weight loss, fever, and clinical symptoms, following challenge with 100 to 1,000 LD50 RPV, and it prevented development of secondary lesions similar to protection seen with NYCBH. Finally, vaccination with either one or two doses of NYCBHΔE3L resulted in similar neutralizing antibody titers following RPV challenge as compared to titers obtained by vaccination with NYCBH. These results support the efficacy of the attenuated NYCBHΔE3L in protection against an orthologous poxvirus challenge.