RESUMO
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.
Assuntos
Vesículas Citoplasmáticas/virologia , Infecções por Enterovirus/transmissão , Enterovirus/fisiologia , Macrófagos/virologia , Vesículas Citoplasmáticas/química , Humanos , Macrófagos/citologia , Fosfatidilserinas , Poliovirus/fisiologia , RNA Viral/metabolismo , Rhinovirus/fisiologia , Replicação ViralRESUMO
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.
Assuntos
Encephalitozoon , Células CACO-2 , Encephalitozoon/genética , Enterócitos , Humanos , Transdução de SinaisRESUMO
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.
Assuntos
Citoplasma/ultraestrutura , Microsporídios/ultraestrutura , Esporos Fúngicos/ultraestrutura , Microscopia Crioeletrônica , Microscopia , Microscopia Eletrônica de Transmissão , Microsporídios/citologia , Esporos Fúngicos/citologiaRESUMO
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.
Assuntos
Anticorpos Antifúngicos/imunologia , Encephalitozoon/genética , Encefalitozoonose/microbiologia , Proteínas Fúngicas/metabolismo , Proteômica , Animais , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Encephalitozoon/imunologia , Encephalitozoon/patogenicidade , Encephalitozoon/ultraestrutura , Encefalitozoonose/patologia , Proteínas Fúngicas/genética , Organelas/metabolismo , Organelas/ultraestrutura , Coelhos , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Proteínas Recombinantes , Esporos Fúngicos/ultraestruturaRESUMO
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.
Assuntos
Gadiformes/parasitologia , Microsporidiose/parasitologia , Pleistophora/ultraestrutura , Animais , Oceano Atlântico , Maine , Microscopia Eletrônica de Transmissão , Músculos/parasitologia , Esporos Fúngicos/ultraestruturaRESUMO
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.
RESUMO
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.
Assuntos
Microsporídios não Classificados/crescimento & desenvolvimento , Microsporídios não Classificados/ultraestrutura , Análise Espaço-Temporal , Coloração e Rotulagem/métodos , Complexo de Golgi/parasitologia , Complexo de Golgi/ultraestrutura , Células HeLa , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Microscopia Confocal , Microscopia de Fluorescência/métodos , Microsporídios não Classificados/fisiologia , Microtúbulos/microbiologia , Esporos Fúngicos/ultraestrutura , Vesículas Transportadoras/microbiologiaRESUMO
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.
Assuntos
Proteínas Fúngicas/genética , Microsporídios não Classificados/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/genética , Albendazol/farmacologia , Sequência de Aminoácidos , Animais , Benzimidazóis/farmacologia , Linhagem Celular , Clonagem Molecular , Sequência Conservada , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/microbiologia , Proteínas Fúngicas/metabolismo , Expressão Gênica , Humanos , Rim , Pulmão , Testes de Sensibilidade Microbiana , Microsporídios não Classificados/genética , Microsporídios não Classificados/metabolismo , Microsporídios não Classificados/ultraestrutura , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
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.
Assuntos
Citoplasma/ultraestrutura , Complexo de Golgi/ultraestrutura , Microsporídios não Classificados/ultraestrutura , Esporos Fúngicos/ultraestrutura , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Microscopia Eletrônica de TransmissãoRESUMO
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.
Assuntos
Microsporídios não Classificados/classificação , Microsporídios não Classificados/patogenicidade , Peixe-Zebra/microbiologia , Animais , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Microscopia , Microsporídios não Classificados/crescimento & desenvolvimento , Microsporídios não Classificados/isolamento & purificação , Dados de Sequência Molecular , Músculo Esquelético/microbiologia , Músculo Esquelético/patologia , Sistema Nervoso/microbiologia , Sistema Nervoso/patologia , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Doenças dos Peixes/microbiologia , Microsporídios/classificação , Microsporídios/isolamento & purificação , Microsporidiose/veterinária , Perciformes/microbiologia , Animais , DNA Fúngico/análise , Feminino , Genes de RNAr , Microscopia Eletrônica de Transmissão , Microsporídios/genética , Microsporídios/fisiologia , Microsporidiose/microbiologia , Dados de Sequência Molecular , Ovário/parasitologia , Ovário/patologia , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie , Esporos Fúngicos/ultraestruturaRESUMO
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.
Assuntos
Encephalitozoon cuniculi/ultraestrutura , Encephalitozoon/ultraestrutura , Esporos Fúngicos/ultraestrutura , Western Blotting , Encephalitozoon/química , Encephalitozoon/metabolismo , Encephalitozoon cuniculi/química , Encephalitozoon cuniculi/metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Microscopia de Fluorescência , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esporos Fúngicos/metabolismo , Vacúolos/metabolismoRESUMO
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.
Assuntos
Encephalitozoon cuniculi/fisiologia , Proteínas Fúngicas/metabolismo , Mapeamento de Interação de Proteínas , Imunoprecipitação , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Organelas/química , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Laringite/diagnóstico , Microsporídios não Classificados/isolamento & purificação , Microsporidiose/diagnóstico , Prega Vocal/patologia , Idoso , Biópsia , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Laringite/microbiologia , Leucemia Linfocítica Crônica de Células B/complicações , Masculino , Microscopia Eletrônica , Microsporídios não Classificados/classificação , Microsporídios não Classificados/ultraestrutura , Microsporidiose/microbiologia , Dados de Sequência Molecular , Micologia/métodos , Filogenia , Análise de Sequência de DNA , Prega Vocal/microbiologiaRESUMO
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.
RESUMO
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.
Assuntos
Infecções por Caliciviridae/transmissão , Vesículas Extracelulares/virologia , Fezes/virologia , Infecções por Rotavirus/transmissão , Animais , Infecções por Caliciviridae/virologia , Pré-Escolar , Transmissão de Doença Infecciosa , Exossomos/virologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos BALB C , Norovirus/genética , Norovirus/patogenicidade , Rotavirus/genética , Rotavirus/patogenicidade , Infecções por Rotavirus/virologia , Suínos , Eliminação de Partículas ViraisRESUMO
The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.
Assuntos
Aquaporinas/análise , Encephalitozoon cuniculi/química , Proteínas Fúngicas/análise , Sequência de Aminoácidos , Animais , Aquaporinas/metabolismo , Células Cultivadas , Dictyostelium , Encefalitozoonose/metabolismo , Proteínas Fúngicas/metabolismo , Glicerol/farmacologia , Humanos , Cloreto de Mercúrio/farmacologia , Oócitos/fisiologia , Osmose/efeitos dos fármacos , Filogenia , Coelhos , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Solventes/farmacologia , Ureia/farmacologia , Xenopus laevis/fisiologiaAssuntos
Cooperação Internacional , Lobosea , Microsporídios , Infecções Oportunistas/microbiologia , Infecções Oportunistas/parasitologia , Animais , Humanos , Lobosea/efeitos dos fármacos , Lobosea/genética , Lobosea/fisiologia , Microsporídios/efeitos dos fármacos , Microsporídios/genética , Microsporídios/fisiologia , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/epidemiologiaRESUMO
Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29 degrees C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and nonactivated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.
Assuntos
Apansporoblastina/crescimento & desenvolvimento , Células Gigantes/citologia , Microsporidiose/fisiopatologia , Esporos Fúngicos/fisiologia , Esporos Fúngicos/ultraestrutura , Animais , Divisão Celular/fisiologia , Linhagem Celular , Células Gigantes/microbiologia , Microscopia Eletrônica de Transmissão , Fagocitose/fisiologia , Coelhos , Fatores de TempoRESUMO
Traditionally, the Microsporidia were primarily studied in insects and fish. There were only a few human cases of microsporidiosis reported until the advent of AIDS, when the number of human microsporidian infections dramatically increased and the importance of these new pathogens to medicine became evident. Over a dozen different kinds of microsporidia infecting humans have been reported. While some of these infections were identified in new genera (Enterocytozoon, Vittaforma), there were also infections identified from established genera such as Pleistophora and Encephalitozoon. The genus Pleistophora, originally erected for a species described from fish muscle, and the genus Encephalitozoon, originally described from disseminated infection in rabbits, suggested a link between human infections and animals. In the 1980's, three Pleistophora sp. infections were described from human skeletal muscle without life cycles presented. Subsequently, the genus Trachipleistophora was established for a human-infecting microsporidium with developmental differences from species of the genus Pleistophora. Thus, the existence of a true Pleistophora sp. or spp. in humans was put into question. We have demonstrated the life-cycle stages of the original Pleistophora sp. infection from human muscle, confirming the existence of a true Pleistophora species in humans, P. ronneafiei Cali et Takvorian, 2003, the first demonstrated in a mammalian host. Another human infection, caused by a parasite from invertebrates, was Brachiola algerae Lowman, Takvorian et Cali, 2000. The developmental stages of this human muscle-infecting microsporidium demonstrate morphologically what we have also confirmed by molecular means, that B. algerae, the mosquito parasite, is the causative agent of this human skeletal muscle infection. B. algerae had previously been demonstrated in humans but only in surface infections, skin and eye. The diagnostic features of B. algerae and P. ronneafiei infections in human skeletal muscle are presented. While Encephalitozoon cuniculi has been known as both an animal (mammal) and human parasite, the idea of human microsporidial infections derived from cold-blooded vertebrates and invertebrates has only been suggested by microsporidian phylogeny based on small subunit ribosomal DNA sequences but has not been appreciated. The morphological data presented here demonstrate these relationships. Additionally, water, as a link that connects microsporidial spores in the environment to potential host organisms, is diagrammatically presented.