Assembly and Function of the Anthrax Toxin Protein Translocation Complex.

Anthrax toxin is a major virulence factor of Bacillus anthracis, a Gram-positive bacterium which can form highly stable spores that are the causative agents of the disease, anthrax. While chiefly a disease of livestock, spores can be "weaponized" as a bio-terrorist agent, and can be deadly if not recognized and treated early with antibiotics. The intracellular pathways affected by the enzymes are broadly understood and are not discussed here. This chapter focuses on what is known about the assembly of secreted toxins on the host cell surface and how the toxin is delivered into the cytosol. The central component is the "Protective Antigen", which self-oligomerizes and forms complexes with its pay-load, either Lethal Factor or Edema Factor. It binds a host receptor, CMG2, or a close relative, triggering receptor-mediated endocytosis, and forms a remarkably elegant yet powerful machine that delivers toxic enzymes into the cytosol, powered only by the pH gradient across the membrane. We now have atomic structures of most of the starting, intermediate and final assemblies in the infectious process. Together with a major body of biophysical, mutational and biochemical work, these studies reveal a remarkable story of both how toxin assembly is choreographed in time and space.

Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, enters a cell through endocytosis, followed by viral and cell membrane fusion. The fusion protein, E1, undergoes an acid pH-induced pre- to postfusion conformation change during membrane fusion. As part of the conformation change, E1 dissociates from the receptor-binding protein, E2, and swivels its domains I and II over domain III to form an extended intermediate and then eventually to form a postfusion hairpin homotrimer. In this study, we tested if the domain I-III linker acts as a "hinge" for the swiveling motion of E1 domains. We found a conserved spring-twisted structure in the linker, stabilized by a salt bridge between a conserved arginine-aspartic acid pair, as a "hinge point" for domain swiveling. Molecular dynamics (MD) simulation of the CHIKV E1 or E2-E1 structure predicted that the spring-twisted region untwists at pH 5.5. Corroborating the prediction, introduction of a "cystine staple" at the hinge point, replacing the conserved arginine-aspartic acid pair with cysteine residues, resulted in loss of fusion activity of E1. MD simulation also predicted domain I-III swiveling at acidic pH. We tested if breaking the His 331-Lys 16 H bond between domains I and III, seen only in the prefusion conformation, is important for domain swiveling. When domains I and III are "stapled" by introducing a disulfide bond in between, E1 showed loss of fusion activity, implying that domain I and III dissociation is a critical acid pH-induced step in membrane fusion. However, replacement of His 331 with an acidic residue did not affect the pH threshold for fusion, suggesting His 331 is not an acid-sensing residue.IMPORTANCEAedes mosquito-transmitted viruses such as the Zika, dengue, and chikungunya viruses have spread globally. CHIKV, similar to many other enveloped viruses, enters cells in sequential steps: step 1 involves receptor binding followed by endocytosis, and step 2 involves viral-cell membrane fusion in the endocytic vesicle. The viral envelope surface protein, E1, performs membrane fusion. E1 is triggered to undergo conformational changes by acidic pH of the maturing endosome. Different domains of E1 rearrange during the pre- to postfusion conformation change. Using in silico analysis of the E1 structure and different biochemical experiments, we explained a structural mechanism of key conformational changes in E1 triggered by acidic pH. We noted two important structural changes in E1 at acidic pH. In the first, a spring-twisted region in a loop connecting two domains (I and III) untwists, bringing a swiveling motion of domains on each other. In the second, breaking of interactions between domains I and III and domain separation are required for membrane fusion. This knowledge will help devise new therapeutic strategies to block conformation changes in E1 and thus viral entry.

Constructing Implantable SrTiO3 :Yb,Ho Nanofibers for NIR-Triggered and Optically Monitored Chemotherapy.

Light-responsive and photoluminescent (PL) drug-delivery platforms have sparked fascinating advancements in personalized tumor chemotherapy due to their unique characteristics in biological imaging and manipulated release kinetics. Herein, implantable Yb3+ and Ho3+ co-doped strontium titanate (SrTiO3 :Yb,Ho) nanofibers were synthesized and decorated on the surface with polyacrylic acid (PAA) molecules. The preliminary in vitro assay confirmed that this implantable fibrous mesh presented sound cytocompatibility. The PAA surface decoration improved the loading capacity of an anticancer drug (doxorubicin (DOX)) and effectively prevented a daunting burst release in a neutral aqueous environment. Owing to the electrostatic bond between PAA and DOX molecules, low-pH microenvironments and NIR (λ=808 nm) irradiation both induced significantly accelerated DOX release and consequently enhanced the local cancer-cell-killing effect. Additionally, the ratio of green-to-red emission (I545 /I655 ) from the SrTiO3 :Yb,Ho-PAA fibers responded effectively to the DOX release progress and dosage due to a fluorescence resonance energy transfer (FRET) effect. This unique characteristic enabled optical monitoring of the delivery progress in a timely manner. These SrTiO3 :Yb,Ho-PAA nanofibers, with precise dual-triggering and optical monitoring of DOX release, are expected to serve as a new implantable drug delivery platform for personalized chemotherapy in the future.