The Function and Structure of the Microsporidia Polar Tube.

Microsporidia are obligate intracellular pathogens that were initially identified about 160 years ago. Current phylogenetic analysis suggests that they are grouped with Cryptomycota as a basal branch or sister group to the fungi. Microsporidia are found worldwide and can infect a wide range of animals from invertebrates to vertebrates, including humans. They are responsible for a variety of diseases once thought to be restricted to immunocompromised patients but also occur in immunocompetent individuals. The small oval spore containing a coiled polar filament, which is part of the extrusion and invasion apparatus that transfers the infective sporoplasm to a new host, is a defining characteristic of all microsporidia. When the spore becomes activated, the polar filament uncoils and undergoes a rapid transition into a hollow tube that will transport the sporoplasm into a new cell. The polar tube has the ability to increase its diameter from approximately 100 nm to over 600 nm to accommodate the passage of an intact sporoplasm and penetrate the plasmalemma of the new host cell. During this process, various polar tube proteins appear to be involved in polar tube attachment to host cell and can interact with host proteins. These various interactions act to promote host cell infection.

Enterocytozoon schreckii n. sp. Infects the Enterocytes of Adult Chinook Salmon (Oncorhynchus tshawytscha) and May Be a Sentinel of Immunosenescence.

A novel Enterocytozoon infection was identified in the intestines of sexually mature Chinook salmon. While microsporidian parasites are common across a diverse range of animal hosts, this novel species is remarkable because it demonstrates biological, pathological, and genetic similarity with Enterocytozoon bieneusi, the most common causative agent of microsporidiosis in AIDS patients. There are similarities in the immune and endocrine processes of sexually mature Pacific salmon and immunocompromised humans, suggesting possible common mechanisms of susceptibility in these two highly divergent host species. The discovery of Enterocytozoon schreckii n. sp. contributes to clarifying the phylogenetic relationships within family Enterocytozoonidae. The phylogenetic and morphological features of this species support the redescription of Enterocytozoon to include Enterospora as a junior synonym. Furthermore, the discovery of this novel parasite may have important implications for conservation, as it could be a sentinel of immune suppression, disease, and prespawning mortality in threatened populations of salmonids. IMPORTANCE In this work, we describe a new microsporidian species that infects the enterocytes of Chinook salmon. This novel pathogen is closely related to Enterocytozoon bieneusi, an opportunistic pathogen commonly found in AIDS patients and other severely immunocompromised humans. The discovery of this novel pathogen is of interest because it has only been found in sexually mature Chinook salmon, which have compromised immune systems due to the stresses of migration and maturation and which share similar pathological features with immunocompromised and senescent humans. The discovery of this novel pathogen could lead to new insights regarding how microsporidiosis relates to immunosuppression across animal hosts.

Human microsporidian pathogen Encephalitozoon intestinalis impinges on enterocyte membrane trafficking and signaling.

Microsporidia are a large phylum of obligate intracellular parasites. Approximately a dozen species of microsporidia infect humans, where they are responsible for a variety of diseases and occasionally death, especially in immunocompromised individuals. To better understand the impact of microsporidia on human cells, we infected human colonic Caco2 cells with Encephalitozoon intestinalis, and showed that these enterocyte cultures can be used to recapitulate the life cycle of the parasite, including the spread of infection with infective spores. Using transmission electron microscopy, we describe this lifecycle and demonstrate nuclear, mitochondrial and microvillar alterations by this pathogen. We also analyzed the transcriptome of infected cells to reveal host cell signaling alterations upon infection. These high-resolution imaging and transcriptional profiling analysis shed light on the impact of the microsporidial infection on its primary human target cell type.This article has an associated First Person interview with the first authors of the paper.

ß-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway.

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.

Invasion of Host Cells by Microsporidia.

Microsporidia are found worldwide and both vertebrates and invertebrates can serve as hosts for these organisms. While microsporidiosis in humans can occur in both immune competent and immune compromised hosts, it has most often been seen in the immune suppressed population, e.g., patients with advanced HIV infection, patients who have had organ transplantation, those undergoing chemotherapy, or patients using other immune suppressive agents. Infection can be associated with either focal infection in a specific organ (e.g., keratoconjunctivitis, cerebritis, or hepatitis) or with disseminated disease. The most common presentation of microsporidiosis being gastrointestinal infection with chronic diarrhea and wasting syndrome. In the setting of advanced HIV infection or other cases of profound immune deficiency microsporidiosis can be extremely debilitating and carries a significant mortality risk. Microsporidia are transmitted as spores which invade host cells by a specialized invasion apparatus the polar tube (PT). This review summarizes recent studies that have provided information on the composition of the spore wall and PT, as well as insights into the mechanism of invasion and interaction of the PT and spore wall with host cells during infection.

An Ultrastructural Study of the Extruded Polar Tube of Anncaliia algerae (Microsporidia).

All microsporidia share a unique, extracellular spore stage, containing the infective sporoplasm and the apparatus for initiating infection. The polar filament/polar tube when exiting the spore transports the sporoplasm through it into a host cell. While universal, these structures and processes have been enigmatic. This study utilized several types of microscopy, describing and extending our understanding of these structures and their functions. Cryogenically preserved polar tubes vary in diameter from 155 to over 200 nm, noticeably larger than fixed-sectioned or negatively stained samples. The polar tube surface is pleated and covered with fine fibrillar material that projects from the surface and is organized in clusters or tufts. These fibrils may be the sites of glycoproteins providing protection and aiding infectivity. The polar tube surface is ridged with 5-6 nm spacing between ridges, enabling the polar tube to rapidly increase its diameter to facilitate the passage of the various cargo including cylinders, sacs or vesicles filled with particulate material and the intact sporoplasm containing a diplokaryon. The lumen of the tube is lined with a membrane that facilitates this passage. Careful examination of the terminus of the tube indicates that it has a closed tip where the membranes for the terminal sac are located.

Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission.

In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.

The role of microsporidian polar tube protein 4 (PTP4) in host cell infection.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

A Novel Fluorescent Labeling Method Enables Monitoring of Spatio-Temporal Dynamics of Developing Microsporidia.

The microsporidium, Anncaliia algerae (Brachiola algerae), is a eukaryotic obligate intracellular parasite first isolated from mosquitoes and is an important opportunistic human pathogen that can cause morbidity and mortality among immune-compromised individuals including patients with AIDS and those undergoing chemotherapy. There is little known about the Microsporidia-host cell interface in living host cells, due to current approaches being limited by the lack of fluorescent reporters for detecting the parasite lifecycle. Here, we have developed and applied novel vital fluorescent parasite labeling methodologies in conjunction with fluorescent protein-tagged reporters to track simultaneously the dynamics of both parasite and host cell specific components, including the secretory and endocytic trafficking pathways, during the entire infection time period. We have found dramatic changes in the dynamics of host secretory trafficking organelles during the course of infection. The Golgi compartment is gradually disassembled and regenerated into mini-Golgi structures in parallel with cellular microtubule depolymerization. Importantly, we find that Microsporidia progeny are associated with these de novo formed mini-Golgi structures. These host structures appear to create a membrane bound niche environment for parasite development. Our studies presented here provide novel imaging tools and methodologies that will facilitate in understanding the biology of microsporidial parasites in the living host.

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Analysis of the beta-tubulin gene and morphological changes of the microsporidium Anncaliia algerae both suggest albendazole sensitivity.

The Microsporidium, Anncaliia algerae, an obligate intracellular parasite, has been identified as an opportunistic human pathogen, but treatment has not been evaluated for infections with this organism. Albendazole, an antitubulin polymerization drug used against parasitic worm infections, has been the medication of choice used to treat some microsporidial infections affecting humans, with varying results ranging from clearing infection (Encephalitozoon) to resistance (Enterocytozoon). This study illustrates the effect of albendazole treatment on A. algerae infection in Rabbit Kidney (RK13) cells and Human Fetal Lung (HFL-1) fibroblasts. Albendazole appears to have an attenuating effect on A. algerae infection and albendazole's IC50 in RK13 cells is 0.1 µg/ml. Long-term treatment inhibits up to 98% of spore production, but interrupting treatment reestablishes the infection without new exposure to the parasite as supported by microscopic observations. The parasite's beta-tubulin gene was purified, cloned, and sequenced. Five of the six specific amino acids, associated with benzimidazole sensitivity, are conserved in A. algerae. These findings suggest that A. algerae is sensitive to albendazole; however, the organism is not completely cleared from cultures.

A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae.

The presence of a new microsporidium is believed to be responsible for an emaciative syndrome observed in farmed gilthead sea bream (Sparus aurata) from different facilities along the Spanish coast. Infected fish were approximately half the average weight and significant mortality was attributed to the condition in some facilities. Clinical signs included anorexia, cachexia and pale internal organs. The microsporidium was found mainly in the intestinal mucosa and occasionally in the submucosa. Morphological, histopathological, ultrastructural and molecular phylogenetic studies were conducted to characterise this organism. This microsporidium undergoes intranuclear development in rodlet cells and enterocytes, and cytoplasmic development mainly in enterocytes and macrophages. The nucleus-infecting plasmodium contains several diplokarya and displays polysporous development which occurs without an interfacial envelope. In the host cell cytoplasm, the parasite develops within a membrane-bound matrix. In both infection locations, the polar tube precursors appear as disks, first with lucent centres, then as fully dense disks as they fuse to form the polar filament, all before division of the plasmodium into sporoblasts. Up to 16 intranuclear spores result from the sporogonic development of a single plasmodium, whereas more than 40 spores result from several asynchronous reproductive cycles in the cytoplasmic infection. Fixed spores are ellipsoidal and diplokaryotic, with five to six coils of an isofilar polar filament in a single row. ssrDNA-based molecular phylogenetic inference places this parasite as a sister clade to crustacean-infecting species of the Enterocytozoonidae and closer to Enterocytozoon bieneusi than to other fish-infecting microsporidians presenting intranuclear development, i.e. Nucleospora, Paranucleospora and Desmozoon. Our studies result in the erection of a new species, Enterospora nucleophila, within the family Enterocytozoonidae, and the description of this family is amended accordingly to accommodate the features of known species assigned to it. Severe histopathological damage occurs in intense infections and this microsporidian is considered a serious emerging threat in sea bream production.

The multilayered interlaced network (MIN) in the sporoplasm of the microsporidium Anncaliia algerae is derived from Golgi.

This study provides evidence for the Golgi-like activity of the multilayered interlaced network (MIN) and new ultrastructural observations of the MIN in the sporoplasm of Anncaliia algerae, a microsporidium that infects both insects and humans. The MIN is attached to the end of the polar tubule upon extrusion from the germinating spore. It surrounds the sporoplasm, immediately below its plasma membrane, and most likely maintains the integrity of the sporoplasm, as it is pulled through the everting polar tube. Furthermore, the MIN appears to deposit its dense contents on the surface of the sporoplasm within minutes of spore discharge thickening the plasma membrane. This thickening is characteristic of the developmental stages of the genus Anncaliia. The current study utilizes transmission electron microscopy (TEM), enzyme histochemistry, and high voltage TEM (HVEM) with 3D tomographic reconstruction to both visualize the structure of the MIN and demonstrate that the MIN is a Golgi-related structure. The presence of developmentally regulated Golgi in the Microsporidia has been previously documented. The current study extends our understanding of the microsporidial Golgi and is consistent with the MIN being involved in the extracellular secretion in Anncaliia algerae. This report further illustrates the unique morphology of the MIN as illustrated by HVEM using 3D tomography.

Ichthyosporidium weissii n. sp. (Microsporidia) infecting the arrow goby (Clevelandia ios).

Gonadal infections by a novel microsporidium were discovered in 34% (13/38) of arrow gobies, Clevelandia ios, sampled over a 3-yr period from Morro Bay Marina in Morro Bay, California. Gonadal tumors had been reported in arrow gobies from this geographic area. The infected gonads, found primarily in females, typically appeared grossly as large, white-gray firm and lobulated masses. Histological examination revealed large, multilobate xenomas within the ovaries and no evidence of neoplasia. Typical of the genus Ichthyosporidium, the large xenomas were filled with developmental stages and pleomorphic spores. Wet mount preparations showed two general spore types: microspores with mean length of 6.2 (7.0-4.9, SD = 0.6, N = 20) µm and mean width of 4.3 (5.3-2.9, SD = 0.8) µm; and less numerous macrospores with mean length of 8.5 (10.1-7.1, SD = 1.0, N = 10) µm and mean width of 5.5 (6.2-4.8, SD = 0.5) µm. Transmission electron microscopy demonstrated stages consistent with the genus and 35-50 turns of the polar filament. Small subunit rDNA gene sequence analysis revealed that the parasite from arrow gobies was most closely related to, but distinct from Ichthyosporidium sp. based on sequences available in GenBank. We conclude that this microsporidium represents a new species of Ichthyosporidium, the first species of this genus described from a member of the family Gobiidae and from the Pacific Ocean.

Development, ultrastructural pathology, and taxonomic revision of the Microsporidial genus, Pseudoloma and its type species Pseudoloma neurophilia, in skeletal muscle and nervous tissue of experimentally infected zebrafish Danio rerio.

The microsporidium Pseudoloma neurophilia was initially reported to infect the central nervous system of zebrafish causing lordosis and eventually death. Subsequently, muscle tissue infections were also identified. To understand the infection process, development, and ultrastructural pathology of this microsporidium, larval and adult zebrafish were fed P. neurophilia spores. Spores were detected in the larval fish digestive tract 3-h postexposure (PE). By 4.5-d PE, developing parasite stages were identified in muscle tissue. Wet preparations of larvae collected at 8-d PE showed aggregates of spores in the spinal cord adjacent to the notochord. All parasite stages, including spores, were present in the musculature of larval fish 8-d PE. Adult zebrafish sacrificed 45-d PE had fully developed infections in nerves. Ultrastructural study of the developmental cycle of P. neurophilia revealed that proliferative stages undergo karyokinesis, producing tetranucleate stages that then divide into uninucleate cells. The plasmalemma of proliferative cells has a previously unreported glycocalyx-like coat that interfaces with the host cell cytoplasm. Sporogonic stages form sporophorous vacuoles (SPOV) derived from the plasmalemmal dense surface coat, which "blisters" off sporonts. Uninucleate sporoblasts and spores develop in the SPOV. The developmental cycle is identical in both nerve and muscle. The SPOV surface is relatively thick and is the outermost parasite surface entity; thus, xenomas are not formed. Based on the new information provided by this study, the taxonomic description of the genus Pseudoloma and its type species, P. neurophilia, is modified and its life cycle described.

Human vocal cord infection with the Microsporidium Anncaliia algerae.

We describe a biopsy proven case of microsporidial infection of the false vocal cords in a 69-yr-old male with a history of chronic lymphocytic leukemia. The patient had hoarseness for several weeks before his admission to the hospital for shortness of breath. He had received chemotherapy with fludarabine 6 wk before this hospital admission. A biopsy of vocal cord nodules demonstrated an organism that was identified as Anncaliia algerae by electron microscopy. Molecular analysis of the small subunit RNA gene amplified by polymerase chain reaction further confirmed the identification of this organism as A. algerae. This case illustrates the ability of this insect pathogen to cause disease in immune-compromised mammalian hosts.

Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.

Interactions of Encephalitozoon cuniculi polar tube proteins.

Microsporidia are eukaryotic, obligate intracellular organisms defined by small spores that contain a single invasion organelle, the polar tube, which coils around the interior of the spore. When these parasites infect host cells, the polar tube is discharged from the anterior pole of the spore, pierces the cell, and transfers sporoplasm into the cytoplasm of the host. Three polar tube proteins (PTP1, PTP2, and PTP3) have been identified in this structure. The interactions of these proteins in the assembly and function of the polar tube are not known. This study was undertaken to examine the protein interactions of the Encephalitozoon cuniculi polar tube proteins (EcPTPs). Immunofluorescence and immunoelectron microscopy confirmed the colocalization of EcPTP1, EcPTP2, and EcPTP3 to the polar tube. Experiments using cross-linkers indicated that EcPTP1, EcPTP2, and EcPTP3 form a complex in the polar tube, which was confirmed by immunoprecipitation using EcPTP1 antiserum. Yeast two-hybrid analysis revealed that full-length EcPTP1, EcPTP2, and EcPTP3 interact with each other in vivo. Both the N and C termini of EcPTP1 were involved in these interactions, but the central region of this protein, which contains a repetitive motif, was not. Further studies of polar tube proteins and their structural interactions may help elucidate the formation of the polar tube during the invasion process.

Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi.

Microsporidia were once considered amitochondriate, but have now been found to retain relict mitochondria called mitosomes. These organelles have been identified by immunolocalization in Trachipleistophora hominis, whereas most data on function have been inferred from the presence of mitochondrial protein-encoding sequences in the genome of Encephalitozoon cuniculi. Here we describe the localization of two such enzymes in E. cuniculi cells. Immunofluorescent localization of ferredoxin involved in mitochondrial iron-sulfur cluster assembly reveals a punctate distribution as expected for mitochondria. In contrast, localization of mitochondrial glycerol-3-phosphate dehydrogenase suggests a cytoplasmic distribution in E. cuniculi and possible relocalization of this typically mitochondrial protein.