RESUMEN
Promyelocytic leukemia nuclear bodies (PM NBs), often referred to as membraneless organelles, are dynamic macromolecular protein complexes composed of a PML protein core and other transient or permanent components. PML NBs have been shown to play a role in a wide variety of cellular processes. This review describes in detail the diverse and complex interactions between small and medium size DNA viruses and PML NBs that have been described to date. The PML NB components that interact with small and medium size DNA viruses include PML protein isoforms, ATRX/Daxx, Sp100, Sp110, HP1, and p53, among others. Interaction between viruses and components of these NBs can result in different outcomes, such as influencing viral genome expression and/or replication or impacting IFN-mediated or apoptotic cell responses to viral infection. We discuss how PML NB components abrogate the ability of adenoviruses or Hepatitis B virus to transcribe and/or replicate their genomes and how papillomaviruses use PML NBs and their components to promote their propagation. Interactions between polyomaviruses and PML NBs that are poorly understood but nevertheless suggest that the NBs can serve as scaffolds for viral replication or assembly are also presented. Furthermore, complex interactions between the HBx protein of hepadnaviruses and several PML NBs-associated proteins are also described. Finally, current but scarce information regarding the interactions of VP3/apoptin of the avian anellovirus with PML NBs is provided. Despite the considerable number of studies that have investigated the functions of the PML NBs in the context of viral infection, gaps in our understanding of the fine interactions between viruses and the very dynamic PML NBs remain. The complexity of the bodies is undoubtedly a great challenge that needs to be further addressed.
Asunto(s)
Virus ADN , Proteínas Nucleares , Adenoviridae , Proteínas Nucleares/metabolismo , Cuerpos Nucleares de la Leucemia Promielocítica , Proteína de la Leucemia Promielocítica/metabolismo , Factores de Transcripción/metabolismo , Virus , Virus ADN/genéticaRESUMEN
The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.
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Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno , Virosis/etiología , Virosis/metabolismo , Actinas/metabolismo , Animales , Citoesqueleto/genética , Regulación Viral de la Expresión Génica , Humanos , Laminas/metabolismo , Membrana Nuclear/metabolismo , Lámina Nuclear/metabolismo , Especificidad de la Especie , Replicación ViralRESUMEN
DNA virus infections are often lifelong and can cause serious diseases in their hosts. Their recognition by the sensors of the innate immune system represents the front line of host defence. Understanding the molecular mechanisms of innate immunity responses is an important prerequisite for the design of effective antivirotics. This review focuses on the present state of knowledge surrounding the mechanisms of viral DNA genome sensing and the main induced pathways of innate immunity responses. The studies that have been performed to date indicate that herpesviruses, adenoviruses, and polyomaviruses are sensed by various DNA sensors. In non-immune cells, STING pathways have been shown to be activated by cGAS, IFI16, DDX41, or DNA-PK. The activation of TLR9 has mainly been described in pDCs and in other immune cells. Importantly, studies on herpesviruses have unveiled novel participants (BRCA1, H2B, or DNA-PK) in the IFI16 sensing pathway. Polyomavirus studies have revealed that, in addition to viral DNA, micronuclei are released into the cytosol due to genotoxic stress. Papillomaviruses, HBV, and HIV have been shown to evade DNA sensing by sophisticated intracellular trafficking, unique cell tropism, and viral or cellular protein actions that prevent or block DNA sensing. Further research is required to fully understand the interplay between viruses and DNA sensors.
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Infecciones por Virus ADN , Herpesviridae , Poliomavirus , ADN Viral/metabolismo , Herpesviridae/genética , Herpesviridae/metabolismo , Humanos , Inmunidad Innata , Poliomavirus/genéticaRESUMEN
The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-ß) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.
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ADN Viral/inmunología , Inmunidad Innata/inmunología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/metabolismo , Infecciones por Polyomavirus/inmunología , Poliomavirus/inmunología , Infecciones Tumorales por Virus/inmunología , Animales , ADN Viral/genética , Interacciones Huésped-Patógeno , Interferón beta/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosforilación , Poliomavirus/genética , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virologíaRESUMEN
The tumorigenic potential of mouse polyomavirus (MPyV) has been studied for decades in cell culture models and has been mainly attributed to nonstructural middle T antigen (MT), which acts as a scaffold signal adaptor, activates Src tyrosine kinases, and possesses transforming ability. We hypothesized that MPyV could also transform mouse cells independent of MT via a Toll-like receptor 4 (TLR4)-mediated inflammatory mechanism. To this end, we investigated the interaction of MPyV with TLR4 in mouse embryonic fibroblasts (MEFs) and 3T6 cells, resulting in secretion of interleukin 6 (IL-6), independent of active viral replication. TLR4 colocalized with MPyV capsid protein VP1 in MEFs. Neither TLR4 activation nor recombinant IL-6 inhibited MPyV replication in MEFs and 3T6 cells. MPyV induced STAT3 phosphorylation through both direct and MT-dependent and indirect and TLR4/IL-6-dependent mechanisms. We demonstrate that uninfected mouse fibroblasts exposed to the cytokine environment from MPyV-infected fibroblasts upregulated the expressions of MCP-1, CCL-5, and α-SMA. Moreover, the cytokine microenvironment increased the invasiveness of MEFs and CT26 carcinoma cells. Collectively, TLR4 recognition of MPyV induces a cytokine environment that promotes the cancer-associated fibroblast (CAF)-like phenotype in noninfected fibroblasts and increases cell invasiveness.
RESUMEN
The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/ß1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin ß1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin ß1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin ß1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.
Asunto(s)
Proteínas de la Cápside/metabolismo , ADN Viral/metabolismo , Carioferinas/metabolismo , Infecciones por Polyomavirus/metabolismo , Infecciones por Polyomavirus/virología , Poliomavirus/fisiología , Sustitución de Aminoácidos , Animales , Transporte Biológico , Proteínas de la Cápside/genética , Línea Celular , Núcleo Celular , Técnica del Anticuerpo Fluorescente , Ratones , Mutación , Señales de Localización Nuclear/genética , Poliomavirus/ultraestructura , Unión Proteica , Ensamble de VirusRESUMEN
The minor structural protein VP2 and its shorter variant, VP3, of mouse polyomavirus (MPyV) are essential for virus exit from the endoplasmic reticulum (ER) during viral trafficking to the nucleus. Here, we followed the role of putative hydrophobic domains (HD) of the minor proteins in membrane affinity and viral infectivity. We prepared variants of VP2, each mutated to decrease hydrophobicity of one of three predicted hydrophobic domains: VP2-mHD1, VP2-mHD2 or VP2-mHD3 mutated in HD1 (amino acids (aa) 60-101), HD2 (aa 125-165) or HD3 (aa 287-307), respectively. Transient production of the mutated proteins revealed that only VP2-mHD2 lost the affinity for intracellular membranes. Cytotoxicity connected with the ability of VP2/VP3 to perforate membranes decreased markedly for VP2-mHD2, but only slightly for VP2-mHD1. The mutant VP2-mHD3 exhibited properties similar to the wild-type protein. MPyV genomes, each carrying one of the mutations, were prepared for virus production. MPyV-mHD1 and MPyV-mHD2 viruses could be isolated, while the HD3 mutation in VP2/VP3 prevented virus assembly. We found that both MPyV-mHD1 and MPyV-mHD2 viruses arrived at the ER without delay and were processed by ER residential enzymes. However, the ability to associate with ER membranes was decreased in the case of MPyV-mHD1 and practically abolished in the case of MPyV-mHD2. Interestingly, while MPyV-mHD2 was not infectious, infection of MPyV-mHD1 virus was delayed. These findings reveal that HD2, common to both VP2 and VP3, is responsible for the membrane binding properties of the minor proteins, while HD1 of VP2 is likely required to stabilize VP2-membrane association and to enhance viral exit from the ER.
Asunto(s)
Proteínas de la Cápside/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Poliomavirus/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Núcleo Celular/metabolismo , Retículo Endoplásmico/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Poliomavirus/genética , Poliomavirus/patogenicidad , Unión Proteica , Dominios ProteicosRESUMEN
INTRODUCTION: Cryptococcus gattii serotype B has been associated with several species of trees in tropical and subtropical regions. Serotype B has been found to be the second most frequently isolated from patients in Colombia, but it has not been isolated from the environment. Therefore, a study was designed to sample plant material from a forest in the province of Cundinamarca, with the aim of establishing the presence and distribution of the C. neoformans complex, specially serotype B. In addition, isolates were evaluated for several phenotypic characteristics associated with virulence. METHODOLOGY: During February and March, 2003, 4 separate sample collections were made. One hundred sixty-seven samples were collected from detritus of eucalyptus and 28 samples from other species of trees. All were processed using the conventional procedure and plated on selective media. The following parameters were measured: cellular and capsular sizes in the filtrates, growth at 37 degrees C, virulence in a mouse animal model system, and mating type as detected by PCR. RESULTS: Cryptococcus gattii serotype B was isolated from 46 eucalyptus samples (27.5%). The cellular size in the filtrates ranged from 3.10 to 4.15 microm and the capsular size from 0.30 and 0.46 microm. The 46 isolates grew at 37 degrees C, but none of them caused the death in the animals during 70-day observation period. All isolates were of mating type a. CONCLUSIONS: This constitutes the first report of the isolation of serotype B, mating type a, from eucalyptus detritus in Colombia.
Asunto(s)
Cryptococcus/aislamiento & purificación , Eucalyptus/microbiología , Serotipificación , Animales , Colombia , Criptococosis/microbiología , Criptococosis/mortalidad , Cryptococcus/clasificación , Cryptococcus/patogenicidad , Microbiología Ambiental , Humanos , Ratones , Fenotipo , Clima TropicalRESUMEN
In 1997, our laboratory reported for the first time the isolation of Cryptococcus neoformans var. gattii serotype C associated with almond tree (Terminalia catappa) detritus. This finding led to a more detailed follow up of the association between the plant and the yeast. Preliminary data have shown that survival of the yeast in almond trees seedlings goes beyond 100 days. The aim of the present study was to establish if under the conditions previously studied, C. neoformans var. gattii would remain viable for longer periods. A total of 83 almond tree seedings, 20-40 cm high, were inoculated with C. neoformans var. gattii serotype C (INS-755). Assays were carried out inoculating the stem or the soil where the seedlings were planted. Observations were undertaken for a period of up to 12 months. As processing techniques we employed the endophytic fungi procedure (stems), maceration (roots, leaves) and standard suspension method (soils). Additionally, microscopic visualization of the yeast in plant tissues was done with trypan blue plus lactophenol. C. neoformans var. gattii was recovered from the inoculated plants for a period of up to 12 months post-inoculation; additionally, the fungus had the capacity to migrate from the stem to the soil and viceversa, without causing macroscopic or microscopic alterations in the plant tissues. This finding suggests that there appears to be an association between the host plant and C. neoformans var. gattii in the environment.
Asunto(s)
Cryptococcus neoformans/fisiología , Terminalia/microbiología , Colorantes , Cryptococcus neoformans/aislamiento & purificación , Ecosistema , Hongos , Interacciones Huésped-Parásitos/fisiología , Hojas de la Planta/microbiología , Hojas de la Planta/fisiología , Terminalia/fisiología , Azul de TripanoRESUMEN
Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model.
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Cryptococcus neoformans/genética , Cryptococcus neoformans/aislamiento & purificación , Animales , Cryptococcus neoformans/clasificación , Cryptococcus neoformans/enzimología , Cryptococcus neoformans/patogenicidad , Ambiente , Masculino , Ratones , Ratones Endogámicos BALB C , Fenotipo , VirulenciaRESUMEN
Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER - a prerequisite for efficient delivery of the viral genome to the nucleus.
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Endocitosis , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Poliomavirus/metabolismo , Animales , Línea Celular , Supervivencia Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Endosomas/metabolismo , Endosomas/virología , RatonesRESUMEN
The interferon (IFN) response, induced as a side effect after transfection of nucleic acids into mammalian cells, is known but inadequately described. We followed the IFN response, the fate of cells, and the possible mechanisms leading to this response in NIH3T3 mouse fibroblasts after DNA nucleofection. The gateway destination vector, phGf, and its derivatives encoding toxic and non-toxic variants of the minor structural proteins of polyomaviruses, VP2 and VP3, were used. DNA vector sequences induced in cells the production of high levels of IFN and the upregulation of the IFN-inducible genes, Mx-1, STAT1, IRF1, and IRF7. The IFN response was not restricted to phGf-derived plasmids. In nucleofected cells, upregulation of the modified γ-histone 2A.X indicating DNA damage and inhibition of cell proliferation were also observed. Although 3T3 cells expressed the Toll-like receptor-9 (TLR9) and vectors used for nucleofection contained unmethylated CpGs, signaling leading to IFN induction was found to be TLR9 independent. However, the early activation of nuclear factor-kappa B suggested the participation of this transcription factor in IFN induction. Surprisingly, in contrast to nucleofection, transfection using a cationic polymer induced only a poor IFN response. Together, the results point to a strong side effect of nucleofection.
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Proliferación Celular , Vectores Genéticos/genética , Interferones/genética , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Islas de CpG/efectos de los fármacos , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/farmacología , Vectores Genéticos/fisiología , Interferones/metabolismo , Ratones , Células 3T3 NIH , Plásmidos/genética , Plásmidos/farmacología , Transfección/métodosRESUMEN
Minor structural proteins of mouse polyomavirus (MPyV) are essential for virus infection. To study their properties and possible contributions to cell death induction, fusion variants of these proteins, created by linking enhanced green fluorescent protein (EGFP) to their C- or N-termini, were prepared and tested in the absence of other MPyV gene products, namely the tumor antigens and the major capsid protein, VP1. The minor proteins linked to EGFP at their C-terminus (VP2-EGFP, VP3-EGFP) were found to display properties similar to their nonfused, wild-type versions: they killed mouse 3T3 cells quickly when expressed individually. Carrying nuclear localization signals at their common C-terminus, the minor capsid proteins were detected in the nucleus. However, a substantial subpopulation of both VP2 and VP3 proteins, as well as of the fusion proteins VP2-EGFP and VP3-EGFP, was detected in the cytoplasm, co-localizing with intracellular membranes. Truncated VP3 protein, composed of 103 C-terminal amino acids, exhibited reduced affinity for intracellular membranes and cytotoxicity. Biochemical studies proved each of the minor proteins to be a very potent inducer of apoptosis, which was dependent on caspase activation. Immuno-electron microscopy showed the minor proteins to be associated with damaged membranes of the endoplasmic reticulum, nuclear envelope and mitochondria as soon as 5 h post-transfection. Analysis of apoptotic markers and cell death kinetics in cells transfected with the wild-type MPyV genome and the genome mutated in both VP2 and VP3 translation start codons revealed that the minor proteins contribute moderately to apoptotic processes in the late phase of infection and both are dispensable for cell destruction at the end of the virus replication cycle.
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Apoptosis/efectos de los fármacos , Proteínas de la Cápside/farmacología , Fibroblastos/virología , Infecciones por Polyomavirus/patología , Poliomavirus , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Ratones , Plásmidos/genética , Factores de TiempoRESUMEN
Introducción. Cryptococcus gattii serotipo B ha sido asociado con diferentes especies de árboles en regiones tropicales y subtropicales. Este serotipo es el segundo de mayor importancia clínica en Colombia, pero no se había recuperado del ambiente en ninguno de los estudios realizados por nuestro grupo. Objetivo. Para establecer la presencia y distribución de las especies del complejo C. neoformans, especialmente del serotipo B, y determinar algunas características fenotípicas relacionadas con la virulencia de los aislamientos recuperados, se diseñó un muestreo de material vegetal en un bosque de piso térmico frío del departamento de Cundinamarca. Materiales y métodos. Durante febrero y marzo de 2003 se realizaron 4 muestreos en el bosque y se tomaron 167 muestras de detritos de eucaliptos y 28 muestras de otras especies de árboles, las cuales se procesaron empleando el método convencional de extracción y siembra en medio selectivo. Se determinó el tamaño celular y capsular en los filtrados, el crecimiento a 37°C, la virulencia en un modelo animal y la pareja sexual por PCR. Resultados. Se aisló C. gattii serotipo B de 46 muestras de eucaliptos (27,5 por ciento ). El tamaño celular en el extracto estuvo entre 3,10 y 4,15 µm y el tamaño capsular entre 0,30 y 0,46 µm. Los 46 aislamientos crecieron a 37°C, pero ninguno de los dos aislamientos empleados causó la muerte de los animales inoculados durante los 70 días de observación. Todos los aislamientos fueron pareja sexual a. Conclusiones. Este estudio es el primer informe sobre el aislamiento de C. gattii, serotipo B, pareja sexual a, en Colombia a partir de detritos de eucaliptos
Asunto(s)
Cryptococcus/aislamiento & purificación , Ecosistema , Eucalyptus/microbiología , Fenotipo , Aves/microbiología , HecesRESUMEN
La criptococosis es causada por las tres variedades de Cryptococcus neoformans, las cuales presentan diferencias fisiológicas y de virulencia, algunas de las cuales se han estudiado para reconocer aspectos de su biología. Este trabajo evaluó las características fenotípicas de aislamientos ambientales de las variedades grubii y gattii, con el fin de establecer diferencias asociadas con el ciclo de vida y la virulencia. Se estudiaron 28 aislamientos serotipo A y 31 serotipo C. Se evaluaron la morfología macroscópica y microscópica de blastoconidias cultivadas en agar Sabouraud y suelos, el crecimiento a 37øC, la producción de 22 enzimas extracelulares, la frecuencia del fenómeno de fructificación haploide, la pareja sexual, el patrón de sensibilidad a toxinas asesinas ( killer) y la virulencia en ratones Balb/c. No se observaron diferencias entre las dos variedades en la morfología macroscópica, la microscópica, ni en el crecimiento a 37ºC ( p>0,05); sin embargo, se observó disminución del tamaño celular y capsular de blastoconidias cultivadas en suelo comparado con el tamaño en Sabouraud ( p<0,05). Adicionalmente, se observó mayor actividad enzimática de proteasas, fosfolipasas, fenoloxidasa y b-glucosidasa en los aislamientos de la variedad grubii al compararlos con los aislamientos de la variedad gattii ( p<0,05). En las dos variedades se observaron estructuras relacionadas con fructificación haploide y todos los aislamientos fueron pareja sexual alfa. Los patrones killer en los aislamientos de la var. grubii fueron el I y el II; en contraste, en la var. gattii se encontraron 7 patrones I, V, IX-XIII. En el modelo animal encontramos que 12 de 22 (54,5 por ciento) aislamientos de la var. grubii causaron la muerte durante el periodo de observación, en tanto que ninguno de los 14 de la var. gattii la causó. La disminución del tamaño de las blastoconidias en suelo y la frecuencia de la pareja alfa con estructuras relacionadas con fructificación haploide sugieren un mecanismo importante para la producción de partículas infecciosas en la naturaleza. Adicionalmente, la mayor actividad enzimática de la var. grubii podría asociarse con su virulencia en el modelo animal.
Asunto(s)
Cryptococcus neoformans , Contaminantes Ambientales , Cryptococcus neoformans , FenotipoRESUMEN
Durante 1996-1997, se llevó a cabo en Cúcuta un estudio para determinar el hábitat de Cryptococcus neoformans var. gattii debido debido a que los casos clínicos ocasionados por esa varidad se presentan con alta frecuencia en dicha ciudad. A partir de 2 de 68 almendros (Terminalia catappa) localizados y estudiados en la ciudad, se logro aislar C. neoformans var gatti serotipo C. El objetivo del presente trabajo fue realizar un seguimiento de los dos almendros positivos, con el fin de evaluar el tiempo de permanencia del hongo en asocio con los detritos en diferentes épocas del año. Se tomaron muestras de los dos árboles durante 26 meses y de 9 almendros adicionales; las muestras se extrajeron con una solución tampón fosfato suplementada con antibióticos; el extracto se sembró en medios selectivos y se identificó la especie con base en la morfología macro y microscópica y en pruebas bioquímicas. La determinación de la variedad y el serotipo se realizó con el empleo de técnicas estandarizadas. Del total de 160 muestras de los dos almendros, 31 (19,3 por ciento) fueron positivas para C. neoformans var gattii serotipo C así como una muestra de 1 de los 9 almendros adicionales. Estos hallazgos confirman que el almendro (Terminalia catappa) pude tener algún papel como planta intermediaria para la var. gattii y, además, que la variedad está presente en el área durante todo el año, ya que se logró recuperar en diferentes meses a lo largo de todo el estudio