RESUMEN
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.
Asunto(s)
Encephalitozoon , Células CACO-2 , Encephalitozoon/genética , Enterocitos , Humanos , Transducción de SeñalRESUMEN
A 12 year-old female spayed felid presented after a 35 day history of right eye pain. On examination, a sub-epithelial opacity was identified in the cornea. A lamellar keratectomy was performed and histopathological analysis revealed low numbers of 2x4um, Gram, Hamatoxylin-eosin and Gomori methanamine-silver positive spores. Transmission electron microscopy found ultrastructural findings consistent with the phylum Microspora. To the author's knowledge, this is only the second case of microsporidial stromal keratitis reported in a felid.
RESUMEN
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.
Asunto(s)
Citoplasma/ultraestructura , Microsporidios/ultraestructura , Esporas Fúngicas/ultraestructura , Microscopía por Crioelectrón , Microscopía , Microscopía Electrónica de Transmisión , Microsporidios/citología , Esporas Fúngicas/citologíaRESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae/transmisión , Vesículas Extracelulares/virología , Heces/virología , Infecciones por Rotavirus/transmisión , Animales , Infecciones por Caliciviridae/virología , Preescolar , Transmisión de Enfermedad Infecciosa , Exosomas/virología , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Norovirus/genética , Norovirus/patogenicidad , Rotavirus/genética , Rotavirus/patogenicidad , Infecciones por Rotavirus/virología , Porcinos , Esparcimiento de VirusRESUMEN
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.
Asunto(s)
Anticuerpos Antifúngicos/inmunología , Encephalitozoon/genética , Encefalitozoonosis/microbiología , Proteínas Fúngicas/metabolismo , Proteómica , Animales , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Encephalitozoon/inmunología , Encephalitozoon/patogenicidad , Encephalitozoon/ultraestructura , Encefalitozoonosis/patología , Proteínas Fúngicas/genética , Orgánulos/metabolismo , Orgánulos/ultraestructura , Conejos , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Proteínas Recombinantes , Esporas Fúngicas/ultraestructuraRESUMEN
Pleistophora macrozoarcidis a microsporidian parasite infecting the muscle tissue of the ocean pout Macrozoarces americanus collected from the Gulf of Maine of the Atlantic Ocean, MA, USA, was morphologically described on the basis of ultrastructural features. Infection was detected as opaque white or rusty brown lesions scattered throughout the musculature of the fish mainly in the region anterior to anus. Transmission electron microscopy showed that in individual parasitized muscle cells, the infection progresses within parasite formed vesicles which are in direct contact with muscle cell elements. The earliest observed parasitic stages are the globular multinucleated proliferative cells or plasmodia limited by a highly tortuous plasmalemma with intervesicular finger-like digitations projecting into the parasite cytoplasm. These cells divided through the invagination of the plasmalemma and the amorphous coat producing daughter-cells. Fine electron-dense secretion is deposited on the plasmalemma that causes its thickening which is a sign of commencement of the sporogonic phase. This phase is carried out by cytokinesis of the sporonts and results in the formation of sporoblasts and finally spores. Mature spore has a thin electron-dense exospore, a thick electron-lucent endospore, and the plasma membrane which encloses the spore contents. A single nucleus is centrally located with the posterior region containing a posterior vacuole. The majority of spores have 7-13 coils in 1-2 rows, and a small group of spores had about 23 coils forming two rows. Events of polar filament extrusion for penetration of uninfected cells were studied. The polaroplast membranes were expanded and occupy most of the length of the spore. The coils are dislocated from the sides of the spore to throughout the entire sporoplasm. The polar filament everts and extrudes through the polar cap with a sufficient force to pierce adjacent sporophorous vesicle walls. After eversion, the polar filament is referred to as a polar tubule, as it forms a tube through which the sporoplasm travels. It pierces anything in its path and deposits the sporoplasm at a new location to begin another infective cycle.
Asunto(s)
Gadiformes/parasitología , Microsporidiosis/parasitología , Pleistophora/ultraestructura , Animales , Océano Atlántico , Maine , Microscopía Electrónica de Transmisión , Músculos/parasitología , Esporas Fúngicas/ultraestructuraRESUMEN
BACKGROUND: White-nose syndrome (WNS) has devastated bat populations in North America, with millions of bats dead. WNS is associated with physiological changes in hibernating bats, leading to increased arousals from hibernation and premature consumption of fat reserves. However, there is evidence of surviving populations of little brown myotis (Myotis lucifugus) close to where the fungus was first detected nearly ten years ago. RESULTS: We examined the hibernation patterns of a surviving population of little brown myotis and compared them to patterns in populations before the arrival of WNS and populations at the peak of WNS mortality. Despite infection with Pseudogymnoascus destructans, the causative fungal agent, the remnant population displayed less frequent arousals from torpor and lower torpid body temperatures than bats that died from WNS during the peak of mortality. The hibernation patterns of the remnant population resembled pre-WNS patterns with some modifications. CONCLUSIONS: These data show that remnant populations of little brown myotis do not experience the increase in periodic arousals from hibernation typified by bats dying from WNS, despite the presence of the fungal pathogen on their skin. These patterns may reflect the use of colder hibernacula microclimates by WNS survivors, and/or may reflect differences in how these bats respond to the disease.
RESUMEN
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.
Asunto(s)
Microsporidia no Clasificados/crecimiento & desarrollo , Microsporidia no Clasificados/ultraestructura , Análisis Espacio-Temporal , Coloración y Etiquetado/métodos , Aparato de Golgi/parasitología , Aparato de Golgi/ultraestructura , Células HeLa , Interacciones Huésped-Parásitos , Humanos , Estadios del Ciclo de Vida , Microscopía Confocal , Microscopía Fluorescente/métodos , Microsporidia no Clasificados/fisiología , Microtúbulos/microbiología , Esporas Fúngicas/ultraestructura , Vesículas Transportadoras/microbiologíaRESUMEN
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.
Asunto(s)
Vesículas Citoplasmáticas/virología , Infecciones por Enterovirus/transmisión , Enterovirus/fisiología , Macrófagos/virología , Vesículas Citoplasmáticas/química , Humanos , Macrófagos/citología , Fosfatidilserinas , Poliovirus/fisiología , ARN Viral/metabolismo , Rhinovirus/fisiología , Replicación ViralRESUMEN
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.
Asunto(s)
Proteínas Fúngicas/genética , Microsporidia no Clasificados/efectos de los fármacos , Esporas Fúngicas/efectos de los fármacos , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/genética , Albendazol/farmacología , Secuencia de Aminoácidos , Animales , Bencimidazoles/farmacología , Línea Celular , Clonación Molecular , Secuencia Conservada , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Fibroblastos/efectos de los fármacos , Fibroblastos/microbiología , Proteínas Fúngicas/metabolismo , Expresión Génica , Humanos , Riñón , Pulmón , Pruebas de Sensibilidad Microbiana , Microsporidia no Clasificados/genética , Microsporidia no Clasificados/metabolismo , Microsporidia no Clasificados/ultraestructura , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Esporas Fúngicas/ultraestructura , Tubulina (Proteína)/metabolismoRESUMEN
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.
Asunto(s)
Apansporoblastina/clasificación , Apansporoblastina/patogenicidad , Enfermedades de los Peces/microbiología , Microsporidiosis/veterinaria , Dorada/microbiología , Animales , Apansporoblastina/genética , Núcleo Celular/microbiología , Núcleo Celular/ultraestructura , Citoplasma/microbiología , Citoplasma/ultraestructura , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Enfermedades de los Peces/patología , Interacciones Huésped-Patógeno , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Microscopía Electrónica de Transmisión , Microsporidiosis/microbiología , Microsporidiosis/patología , Datos de Secuencia Molecular , FilogeniaRESUMEN
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.
Asunto(s)
Citoplasma/ultraestructura , Aparato de Golgi/ultraestructura , Microsporidia no Clasificados/ultraestructura , Esporas Fúngicas/ultraestructura , Tomografía con Microscopio Electrónico , Imagenología Tridimensional , Microscopía Electrónica de TransmisiónRESUMEN
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.
Asunto(s)
Enfermedades de los Peces/microbiología , Microsporidios/clasificación , Microsporidios/aislamiento & purificación , Microsporidiosis/veterinaria , Perciformes/microbiología , Animales , ADN de Hongos/análisis , Femenino , Genes de ARNr , Microscopía Electrónica de Transmisión , Microsporidios/genética , Microsporidios/fisiología , Microsporidiosis/microbiología , Datos de Secuencia Molecular , Ovario/parasitología , Ovario/patología , Filogenia , Análisis de Secuencia de ADN , Especificidad de la Especie , Esporas Fúngicas/ultraestructuraRESUMEN
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.
Asunto(s)
Microsporidia no Clasificados/clasificación , Microsporidia no Clasificados/patogenicidad , Pez Cebra/microbiología , Animales , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Microscopía , Microsporidia no Clasificados/crecimiento & desarrollo , Microsporidia no Clasificados/aislamiento & purificación , Datos de Secuencia Molecular , Músculo Esquelético/microbiología , Músculo Esquelético/patología , Sistema Nervioso/microbiología , Sistema Nervioso/patología , ARN de Hongos/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADNRESUMEN
The microsporidia are emerging human and veterinary pathogens known to infect every tissue type and organ system. Their infectious spore possesses a number of peculiar organelles, including the diagnostic polar tube. In a proteomics-driven effort to find novel components of this organelle in the human-pathogenic species Encephalitozoon cuniculi, we unexpectedly discovered a protein which localizes to punctate structures consistent with the appearance of relic mitochondria, or mitosomes. However, this novel protein did not colocalize with ferredoxin, a mitochondrial iron-sulfur cluster protein which shows a similar localization pattern by light microscopy. The distribution pattern of this protein thus suggests either a novel vesicular compartment that is similar to mitosomes in size and distribution, the presence of subdomains or branching architecture within mitosomes, or heterogeneity in the protein composition of E. cuniculi mitosomes.
Asunto(s)
Vesículas Citoplasmáticas/química , Encephalitozoon cuniculi/química , Secuencia de Aminoácidos , Anticuerpos/inmunología , Vesículas Citoplasmáticas/inmunología , Encephalitozoon cuniculi/genética , Encephalitozoon cuniculi/inmunología , Humanos , Datos de Secuencia Molecular , Transporte de Proteínas , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Alineación de SecuenciaRESUMEN
The microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles in Encephalitozoon cuniculi. As a result, a new component of the E. cuniculi developing spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole of Encephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.
Asunto(s)
Encephalitozoon cuniculi/ultraestructura , Encephalitozoon/ultraestructura , Esporas Fúngicas/ultraestructura , Western Blotting , Encephalitozoon/química , Encephalitozoon/metabolismo , Encephalitozoon cuniculi/química , Encephalitozoon cuniculi/metabolismo , Proteínas Fúngicas/análisis , Proteínas Fúngicas/metabolismo , Microscopía Fluorescente , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esporas Fúngicas/metabolismo , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
Laringitis/diagnóstico , Microsporidia no Clasificados/aislamiento & purificación , Microsporidiosis/diagnóstico , Pliegues Vocales/patología , Anciano , Biopsia , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Humanos , Laringitis/microbiología , Leucemia Linfocítica Crónica de Células B/complicaciones , Masculino , Microscopía Electrónica , Microsporidia no Clasificados/clasificación , Microsporidia no Clasificados/ultraestructura , Microsporidiosis/microbiología , Datos de Secuencia Molecular , Micología/métodos , Filogenia , Análisis de Secuencia de ADN , Pliegues Vocales/microbiologíaRESUMEN
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.
Asunto(s)
Encephalitozoon cuniculi/fisiología , Proteínas Fúngicas/metabolismo , Mapeo de Interacción de Proteínas , Inmunoprecipitación , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Orgánulos/química , Unión Proteica , Técnicas del Sistema de Dos HíbridosAsunto(s)
Cooperación Internacional , Lobosea , Microsporidios , Infecciones Oportunistas/microbiología , Infecciones Oportunistas/parasitología , Animales , Humanos , Lobosea/efectos de los fármacos , Lobosea/genética , Lobosea/fisiología , Microsporidios/efectos de los fármacos , Microsporidios/genética , Microsporidios/fisiología , Infecciones Oportunistas/tratamiento farmacológico , Infecciones Oportunistas/epidemiologíaRESUMEN
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.