RESUMO
Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction.While there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus infected primary neurons, grown on plastic coverslips, to allow for sectioning of neurons and axons in their growth plane. This technique allows for TEM examination of cell bodies, axons, growth cones and varicosities, providing powerful insights into virus-cell interaction.
Assuntos
Herpes Simples , Herpesvirus Humano 1 , Microscopia Eletrônica de Transmissão , Neurônios , Herpes Simples/metabolismo , Herpes Simples/patologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Humanos , Neurônios/metabolismo , Neurônios/ultraestrutura , Neurônios/virologiaRESUMO
Transmission immunoelectron microscopy allows for the ultrastructural detection and localization of herpes simplex virus-1 (HSV-1) particles and viral proteins within the infected cell and their relation to the cell cytoskeleton, cellular proteins, vesicles, membranes, and organelles. For the successful application of immunoelectron microscopy, preservation of cell ultrastructure and of epitope antigenicity is essential during sample preparation. This chapter describes the use of chemical fixation followed by rapid cooling of HSV-1 infected sensory neurons in the presence of sucrose as a cryoprotectant to achieve optimal preservation of cell morphology and the use of freeze substitution and resin polymerization at low temperatures for preservation of protein antigenicity. In order to examine HSV-1 infection in the specialized compartments of the neurons (cell body, axons, and growth cones), neurons cultured on plastic coverslips are flat embedded prior to resin polymerization. Overall, this method allows for the ultrathin sectioning and immunogold labeling of the neurons and their axons in growth plane.
Assuntos
Gânglios Espinais , Herpes Simples , Herpesvirus Humano 1 , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Neurônios , Animais , Galinhas , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Gânglios Espinais/virologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/ultraestrutura , Neurônios/virologia , RatosRESUMO
PURPOSE: In crescentic glomerulonephritis (CGN), the development of cellular bridges between podocytes and parietal epithelial cells (PECs) triggers glomerular crescent formation. However, the sequential changes in glomerular ultrastructure in CGN are not well defined. This study investigated the time course of glomerular ultrastructure in experimental CGN. METHODS: Transmission electron microscopy (TEM) was performed using kidney samples from rats with nephrotoxic serum nephritis (NSN) from day 1 to day 14. Morphometric analysis was conducted on randomly selected glomeruli captured on TEM digital images. RESULTS: On day 1 of NSN, there was widespread formation of focal contacts between the cell bodies of neighboring podocytes, and tight junctions were evident at the site of cell-to-cell contact. This was confirmed by the increased expression of the tight junction molecule, zonula occludens-1 (ZO-1), which localized to the points of podocyte cell-cell body contact. On day 2, the interpodocyte distance decreased and the glomerular basement membrane thickness increased. Foot process effacement (FPE) was segmental on day 3 and diffuse by day 5, accompanied by the formation of podocyte cellular bridges with Bowman's capsule, as confirmed by a decrease in podocyte-to-PEC distance. Fibrinoid necrosis and cellular crescents were evident in all glomeruli by days 7 and 14. In vitro, the exposure of podocytes to macrophage-conditioned media altered cellular morphology and caused an intracellular redistribution of ZO-1. CONCLUSION: The formation of tight junctions between podocytes is an early ultrastructural abnormality in CGN, preceding FPE and podocyte bridge formation and occurring in response to inflammatory injury. Podocyte-to-podocyte tight junction formation may be a compensatory mechanism to maintain the integrity of the glomerular filtration barrier following severe endocapillary injury.
RESUMO
UNLABELLED: The herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9(-)), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (â¼90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD, and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40 to 75%) in varicosities and growth cones infected with strain 17, F, and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with the KOS strain virus (which also lacks pUS9) and still lower (<2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, first in anterograde axonal transport and second in virus assembly in growth cones in distal axons. IMPORTANCE: HSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal cell body are not impaired in the absence of pUS9. Thus, our findings indicate that pUS9 contributes to the overall HSV-1 anterograde axonal transport, including a major role in virus assembly at the axon terminus, which is not essential in the neuronal cell body. Overall, our data suggest that the process of virus assembly at the growth cones differs from that in the neuronal cell body and that HSV-1 has evolved different mechanisms for virus assembly and exit from different cellular compartments.
Assuntos
Transporte Axonal , Cones de Crescimento/virologia , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Lipoproteínas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Gânglios Espinais/citologia , Gânglios Espinais/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Técnicas de Cultura de Órgãos , Ratos WistarRESUMO
Cytokinesis is the final stage of cell division and produces two independent daughter cells. Vesicles derived from internal membrane stores, such as the Golgi, lysosomes, and early and recycling endosomes accumulate at the intracellular bridge (ICB) during cytokinesis. Here, we use electron tomography to show that many ICB vesicles are not independent but connected, forming a newly described ICB vesicular structure - narrow tubules that are often branched. These 'midbody tubules' labelled with horseradish peroxidase (HRP) within 10 min after addition to the surrounding medium demonstrating that they are derived from endocytosis. HRP-labelled vesicles and tubules were observed at the rim of the ICB after only 1 min, suggesting that midbody tubules are likely to be generated by local endocytosis occurring at the ICB rim. Indeed, at least one tubule was open to the extracellular space, indicative of a local origin within the ICB. Inhibition of cholesterol-dependent endocytosis by exposure to methyl-ß-cyclodextrin and filipin reduced formation of HRP-labelled midbody tubules, and induced multinucleation following ICB formation. In contrast, dynamin inhibitors, which block clathrin-mediated endocytosis, induced multinucleation but had no effect on the formation of HRP-labelled midbody tubules. Therefore, our data reveal the existence of a cholesterol-dependent endocytic pathway occurring locally at the ICB, which contributes to the accumulation of vesicles and tubules that contribute to the completion of cytokinesis.
Assuntos
Colesterol/metabolismo , Citocinese/fisiologia , Endocitose/fisiologia , Endossomos/metabolismo , Lisossomos/metabolismo , Complexo de Golgi/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Microscopia Eletrônica/métodos , beta-Ciclodextrinas/metabolismoRESUMO
BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function.
Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citoplasma/genética , Quebras de DNA , Feminino , Humanos , Células MCF-7 , Mitocôndrias/genética , Mitocôndrias/patologia , Estrutura Terciária de Proteína , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Deleção de Sequência , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. MATERIALS AND METHODS: Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. RESULTS: Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 µg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 µg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 µg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. CONCLUSIONS: Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis.
Assuntos
Arecolina/toxicidade , Agonistas Colinérgicos/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Autofagia , Contagem de Células , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Corantes , Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , L-Lactato Desidrogenase/análise , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Mitocôndrias/efeitos dos fármacos , Tamanho Mitocondrial/efeitos dos fármacos , Necrose , Fibrose Oral Submucosa/patologia , Ribossomos/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Vacúolos/efeitos dos fármacosRESUMO
Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction. Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus-cell interaction.
Assuntos
Axônios/ultraestrutura , Biologia Molecular/métodos , Neurônios/ultraestrutura , Simplexvirus/genética , Axônios/patologia , Axônios/virologia , Herpes Simples/diagnóstico , Herpes Simples/patologia , Herpes Simples/virologia , Humanos , Microscopia Eletrônica de Transmissão , Neurônios/patologia , Neurônios/virologia , Cultura Primária de Células , Simplexvirus/metabolismoRESUMO
Mutations in serine palmitoyltransferase long chain subunit 1 (SPTLC1) cause the typical length-dependent axonal degeneration hereditary sensory neuropathy type 1 (HSN1). Transmission electron microscopy studies on SPTLC1 mutant lymphoblasts derived from patients revealed specific structural abnormalities of mitochondria. Swollen mitochondria with abnormal cristae were clustered around the nucleus, with some mitochondria being wrapped in rough endoplasmic reticulum (ER) membranes. Total mitochondrial counts revealed a significant change in mitochondrial numbers between healthy and diseased lymphocytes but did not reveal any change in length to width ratios nor were there any changes to cellular function. However, there was a notable change in ER homeostasis, as assessed using key ER stress markers, BiP and ERO1-Lα, displaying reduced protein expression. The observations suggest that SPTLC1 mutations cause mitochondrial abnormalities and ER stress in HSN1 cells.
Assuntos
Estresse do Retículo Endoplasmático/genética , Linfócitos/patologia , Mitocôndrias/química , Mitocôndrias/genética , Serina C-Palmitoiltransferase/genética , Western Blotting , Citometria de Fluxo , Humanos , Linfócitos/ultraestrutura , Microscopia Confocal , Mitocôndrias/ultraestrutura , Mutação/genéticaRESUMO
Lipotoxicity is an overload of lipids in non-adipose tissues that affects function and induces cell death. Lipotoxicity has been demonstrated in bone cells in vitro using osteoblasts and adipocytes in coculture. In this condition, lipotoxicity was induced by high levels of saturated fatty acids (mostly palmitate) secreted by cultured adipocytes acting in a paracrine manner. In the present study, we aimed to identify the underlying mechanisms of lipotoxicity in human osteoblasts. Palmitate induced autophagy in cultured osteoblasts, which was preceded by the activation of autophagosomes that surround palmitate droplets. Palmitate also induced apoptosis though the activation of the Fas/Jun kinase (JNK) apoptotic pathway. In addition, osteoblasts could be protected from lipotoxicity by inhibiting autophagy with the phosphoinositide kinase inhibitor 3-methyladenine or by inhibiting apoptosis with the JNK inhibitor SP600125. In summary, we have identified two major molecular mechanisms of lipotoxicity in osteoblasts and in doing so we have identified a new potential therapeutic approach to prevent osteoblast dysfunction and death, which are common features of age-related bone loss and osteoporosis.
RESUMO
Herpes simplex virus 1 (HSV-1) enters neurons primarily by fusion of the viral envelope with the host cell plasma membrane, leading to the release of the capsid into the cytosol. The capsid travels via microtubule-mediated retrograde transport to the nuclear membrane, where the viral DNA is released for replication in the nucleus. In the present study, the composition and kinetics of incoming HSV-1 capsids during entry and retrograde transport in axons of human fetal and dissociated rat dorsal root ganglia (DRG) neurons were examined by wide-field deconvolution microscopy and transmission immunoelectron microscopy (TIEM). We show that HSV-1 tegument proteins, including VP16, VP22, most pUL37, and some pUL36, dissociated from the incoming virions. The inner tegument proteins, including pUL36 and some pUL37, remained associated with the capsid during virus entry and transit to the nucleus in the neuronal cell body. By TIEM, a progressive loss of tegument proteins, including VP16, VP22, most pUL37, and some pUL36, was observed, with most of the tegument dissociating at the plasma membrane of the axons and the neuronal cell body. Further dissociation occurred within the axons and the cytosol as the capsids moved to the nucleus, resulting in the release of free tegument proteins, especially VP16, VP22, pUL37, and some pUL36, into the cytosol. This study elucidates ultrastructurally the composition of HSV-1 capsids that encounter the microtubules in the core of human axons and the complement of free tegument proteins released into the cytosol during virus entry.
Assuntos
Gânglios Espinais/virologia , Herpesvirus Humano 1/fisiologia , Neurônios/virologia , Proteínas Estruturais Virais/metabolismo , Internalização do Vírus , Animais , Células Cultivadas , Herpesvirus Humano 1/ultraestrutura , Humanos , Microscopia , Ratos , Ratos Wistar , Proteínas Estruturais Virais/ultraestrutura , Vírion/ultraestruturaAssuntos
Glomerulonefrite Membranoproliferativa/patologia , Corpos de Inclusão/ultraestrutura , Glomérulos Renais/ultraestrutura , Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/complicações , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/complicações , Colangiocarcinoma/tratamento farmacológico , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Glomerulonefrite Membranoproliferativa/complicações , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , GencitabinaRESUMO
In Malaysia, co-circulation of CRF01_AE and subtype B has resulted in the emergence of the second generation derivative; CRF33_01B in approximately 20% of its HIV-1 infected individuals. Our objective was to identify possible biological advantages that CRF33_01B possesses over its progenitors. Biological and molecular comparisons of CRF33_01B against its parental subtypes clearly show that CRF33_01B replicated better in activated whole peripheral blood mononuclear cells (PBMCs) and CD4+ T-lymphocytes, but not monocyte-derived macrophages (MDMs). Also, its acquired fitness was greater than CRF01_AE but not subtype B. Moreover, CRF33_01B has higher rate of apoptotic cell death and syncytia induction compared to subtype B. These adaptive and survival abilities could have been acquired by CRF33_01B due to the incorporation of subtype B fragments into the gag-RT region of its full-length genome. Our studies confirm the previously held belief that HIV-1 strains may harbor enhanced biological fitness upon recombination. We therefore estimate a possible gradual replacement of the current predominance of CRF01_AE, as well as wider dissemination of CRF33_01B, together with the identification of other new CRF01_AE/B inter-subtype recombinants in Malaysia.
Assuntos
Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/patogenicidade , Apoptose , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Genótipo , Células Gigantes , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Leucócitos Mononucleares/virologia , Macrófagos/virologia , Malásia/epidemiologia , Recombinação Genética , Replicação ViralRESUMO
Glioneuronal tumors of the central nervous system (CNS) comprise a group of generally low-grade tumors expressing glial and neuronal cells of varying differentiation. Papillary glioneuronal tumor (PGNT) is a new tumor identified in the World Health Organization's classification of CNS tumors (2007). We report a patient with PGNT and highlight the diagnostic features, including a description of "spidery" astrocytes.
Assuntos
Neoplasias Encefálicas/patologia , Lobo Frontal/patologia , Ganglioglioma/patologia , Adolescente , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Craniotomia , Feminino , Ganglioglioma/metabolismo , Ganglioglioma/cirurgia , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Microscopia Eletrônica de TransmissãoRESUMO
Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.
Assuntos
Axônios/virologia , Exocitose , Cones de Crescimento/virologia , Herpesvirus Humano 1/fisiologia , Vesículas Secretórias/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Axônios/ultraestrutura , Linhagem Celular , Proteína GAP-43/análise , Cones de Crescimento/ultraestrutura , Humanos , Cinesinas/análise , Microscopia Imunoeletrônica , Vesículas Secretórias/química , Proteína 25 Associada a Sinaptossoma/análise , Proteína rab3A de Ligação ao GTP/análiseRESUMO
Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Deltaplc1 and Deltaplc2 deletion mutants. In Deltaplc1, which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37 degrees C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Deltaplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Deltaplc2, which was as virulent as WT, Deltaplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25 degrees C, demonstrating that mechanism(s) independent of the 37 degrees C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.
Assuntos
Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/patogenicidade , Fosfoinositídeo Fosfolipase C/fisiologia , Animais , Caenorhabditis elegans/microbiologia , Criptococose/microbiologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Modelos Animais de Doenças , Farmacorresistência Fúngica/genética , Estrenos/farmacologia , Proteínas Fúngicas/metabolismo , Deleção de Genes , Marcação de Genes , Genes Fúngicos , Melaninas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosfoinositídeo Fosfolipase C/antagonistas & inibidores , Fosfoinositídeo Fosfolipase C/genética , Transporte Proteico , Pirrolidinonas/farmacologia , Virulência/genéticaRESUMO
Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.
Assuntos
Parede Celular/metabolismo , Cryptococcus neoformans/enzimologia , Lisofosfolipase/fisiologia , Motivos de Aminoácidos , Transporte Biológico , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Lisofosfolipase/química , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Frações Subcelulares , Superóxido Dismutase/metabolismo , TemperaturaRESUMO
The development of analytical techniques for the measurement of trace elements in cellular compartments of developing teeth remains an important methodological issue in dental research. Recent advances in third generation synchrotron facilities have provided high brilliance X-ray sources that can be effectively used to study trace element distributions in small spatial regions with low detection limits. The present study describes for the first time the application of synchrotron radiation induced X-ray emission (SRIXE) in measuring the distribution of zinc and lead in the ameloblasts of developing Wistar rat teeth. Wistar rats were fed a standard rat diet, containing the normal dietary requirements of zinc, ad libitum and exposed to 100 ppm of lead in drinking water. Resin embedded sections of first mandibular molars were analysed using a 13.3 keV incident monochromatic X-ray beam focussed to a 0.2 microm spot. Characteristic X-rays arising from the entire thickness of the sample were measured using an energy dispersive detector for quantitative analysis of elemental concentrations. The results showed that intranuclear concentrations of zinc were greater than levels in the cytoplasm. Furthermore, nuclear and cytoplasmic concentrations of zinc in the maturation stage (742+/-27 and 424+/-25 ppm, respectively) were significantly higher than the zinc levels observed in the nucleus and cytoplasm of presecretory stage ameloblasts (132+/-10 and 109+/-10 ppm, respectively) (p<0.05). A clear lead signal above the background was not detected in the ameloblasts and lead concentrations could only be reliably measured in the developing enamel. Overall, SRIXE was an effective method of studying the spatial distribution of zinc in the cells of developing teeth and offered a unique combination of sub-micron spatial resolution and parts-per-million detection limits (0.8-1 and 0.6-1 ppm for zinc and lead, respectively).
Assuntos
Ameloblastos/metabolismo , Microanálise por Sonda Eletrônica/métodos , Chumbo/análise , Zinco/análise , Ameloblastos/ultraestrutura , Animais , Células Cultivadas , Microanálise por Sonda Eletrônica/normas , Feminino , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Varicella-zoster virus (VZV) is a species-specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and nonneuronal cells. Enveloped virions were visualized by transmission electron microscopy in neurons and nonneuronal cells and within the extracellular space. Moreover, rather than remaining highly cell associated during infection of cultured cells, such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG.