Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (~400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (~1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Author SummaryWhile the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.National Institutes of Health (AI048082, AI44070, GM31318, RR1088

CRISPR interference interrogation of COPD GWAS genes reveals the functional significance of desmoplakin in iPSC-derived alveolar epithelial cells

Genome-wide association studies (GWAS) have identified dozens of loci associated with chronic obstructive pulmonary disease (COPD) susceptibility; however, the function of associated genes in the cell type(s) affected in disease remains poorly understood, partly due to a lack of cell models that recapitulate human alveolar biology. Here, we apply CRISPR interference to interrogate the function of nine genes implicated in COPD by GWAS in induced pluripotent stem cell–derived type 2 alveolar epithelial cells (iAT2s). We find that multiple genes implicated by GWAS affect iAT2 function, including differentiation potential, maturation, and/or proliferation. Detailed characterization of the GWAS gene DSP demonstrates that it regulates iAT2 cell-cell junctions, proliferation, mitochondrial function, and response to cigarette smoke–induced injury. Our approach thus elucidates the biological function, as well as disease-relevant consequences of dysfunction, of genes implicated in COPD by GWAS in type 2 alveolar epithelial cells.This work was supported by a CJ Martin Early Career Fellowship from the Australian National Health and Medical Research Council awarded to R.B.W.; NIH grant F30HL147426 awarded to K.M.A.; NIH grants U01TR001810, R01DK101501, and R01DK117940 awarded to A.A.W.; NIH grants R01HL135142, R01HL137927, and R01HL147148 awarded to M.H.C.; and NIH grants R01HL127200 and R01HL148667 awarded to X.Z

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins)

Recombinant Lloviu virus as a tool to study viral replication and host responses

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Cryo-EM and molecular docking shows myosin-S1 loop 4 contacts actin and tropomyosin on thin filaments

The motor protein, myosin, drives muscle and non-muscle motility by binding to and moving along actin of thin filaments. Myosin-binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin-binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding non-muscle cell motility. Indeed, experimental approaches, such as fiber diffraction, cryo-electron microscopy and 3D reconstruction, have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryo-EM reconstruction of myosin-S1‒decorated F-actin-tropomyosin together with atomic-scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39 to 42 residue-long tropomyosin pseudo-repeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation

The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes