The biogenesis and dynamics of cellular membranes are governed by fusion and fission processes that ensure the maintenance of closed compartments. These principles also apply to viruses during ...acquisition of their envelope. Based on conventional electron microscopy (EM), however, it has been proposed that poxviruses assemble from membranes made de novo with "free" ends in the cytoplasm. Here, we analyze the origin and structure of poxvirus membranes in a close-to-native state and in three dimensions by using cryopreservation and electron tomography (ET). By cryo-EM, the precursor membrane of poxviruses appears as an open membrane sheet stabilized by a protein scaffold. ET shows that this membrane is derived from pre-existing cellular membranes that rupture to generate an open compartment, rather than being made de novo. Thus, poxvirus infection represents an excellent system to study how cytoplasmic membranes can form open sheets by a process distinct from well-defined mechanisms of membrane biogenesis.
Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is ...known. Here we show that during fast, stop-and-go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane-associated filaments. While the spatial organization of the large parasite organelles appears dependent on their linkage to the cortex, the specialized secretory vesicles are mostly not linked to microtubules or other cellular structures that could provide support for movement.
Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide‐distributed zoonosis. New effective vaccines against CE could potentially have great ...economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix‐assisted laser desorption/ionization‐TOF/TOF. Resulting peptides were termed “clean linear B cell epitopes,” and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D‐modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.
Proteomic analysis of tegumental antigens from Echinococcus granulosus was performed resulting in 58 proteins, 22 of them being described here for the first time. Then, through an innovative bioimmunoinformatic workflow, nine of them were selected as novel protein vaccine candidates. Additionally, through prediction of linear B cell epitopes and mapping within matrix‐assisted laser desorption/ionization (MALDI)‐TOF/TOF peptides, 14 epitopes were selected as peptide‐based vaccine candidates
During intraerythrocytic development, Plasmodium falciparum increases the ion permeability of the erythrocyte plasma membrane to an extent that jeopardizes the osmotic stability of the host cell. A ...previously formulated numeric model has suggested that the parasite prevents premature rupture of the host cell by consuming hemoglobin (Hb) in excess of its own anabolic needs. Here, we have tested the colloid‐osmotic model on the grounds of time‐resolved experimental measurements on cell surface area and volume. We have further verified whether the colloid‐osmotic model can predict time‐dependent volumetric changes when parasites are grown in erythrocytes containing the hemoglobin variants S or C. A good agreement between model‐predicted and empirical data on both infected erythrocyte and intracellular parasite volume was found for parasitized HbAA and HbAC erythrocytes. However, a delayed induction of the new permeation pathways needed to be taken into consideration for the latter case. For parasitized HbAS erythrocyte, volumes diverged from model predictions, and infected erythrocytes showed excessive vesiculation during the replication cycle. We conclude that the colloid‐osmotic model provides a plausible and experimentally supported explanation of the volume expansion and osmotic stability of P. falciparum‐infected erythrocytes. The contribution of vesiculation to the malaria‐protective function of hemoglobin S is discussed.
Summary
Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent‐shaped sporozoites move on 2D ...substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non‐motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo‐electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As aconsequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.
Many intracellular pathogens remodel the actin of their host cells, and the human malaria parasite Plasmodium falciparum is no exception to this rule. The surprising finding is that several ...hemoglobinopathies that protect carriers from severe malaria may do so by interfering with host actin reorganization. Here we discuss our current understanding of actin remodeling in P. falciparum-infected erythrocytes, how hemoglobinopathies interfere with this process, and how impaired host actin remodeling affects the virulence of P. falciparum.
Summary
Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry ...have been limited to insights from non‐human parasites, with no studies at sub‐micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub‐cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three‐dimensional study of invasion using electron microscopy, cryo‐electron tomography and cryo‐X‐ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.
Invasion of the human erythrocyte by Plasmodium parasites is critical for establishment of malaria disease. Over several decades researchers have developed a detailed molecular map of events underpinning invasion, however, cellular understanding of entry by P. falciparum – the parasite responsible for most mortality from malaria – has remained limited. In this issue, Hanssen and Dekiwadia et al. address this shortcoming, presenting the first 3D study of invasion using state‐of‐the‐art electron microscopy to reveal high‐definition insights into this key step in parasite biology.
In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and ...extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail.
The three-dimensional structure of adenovirus has been determined by image reconstruction from cryo-electron micrographs. Comparison with the high resolution X-ray crystal structure of hexon, the ...major capsid protein, enabled an unusually detailed interpretation of the density map and confirmed the validity of the reconstruction. The hexon packing in the capsid shows more extensive intermolecular interfaces between facets than previously proposed. The reconstruction provides the first three-dimensional visualization of the vertex proteins, including the penton base and its associated protruding fiber. Three minor capsid proteins that stabilize and modulate capsomer interactions are revealed. One of these components stabilizes the group-of-nine hexons in the center of each facet and the other two bridge hexons in adjacent facets. The strategic positions of these proteins highlight the importance of cementing proteins in stabilizing a complex assembly.
Proliferation of
in red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms,
replicates by multiple rounds ...of nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of critical nuclear division factors remain poorly understood. Centriolar plaques, the centrosomes of
, serve as microtubule organizing centers and have an acentriolar, amorphous structure. The small size of parasite nuclei has precluded detailed analysis of intranuclear microtubule organization by classical fluorescence microscopy. We apply recently developed super-resolution and time-lapse imaging protocols to describe microtubule reconfiguration during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals two distinct compartments of the centriolar plaque. Whereas centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin but protein-dense. This study generates a working model for an unconventional centrosome and enables a better understanding about the diversity of eukaryotic cell division.
PDF
