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At least 19 recordsLinked to original sources
PubMed2002

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation.

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.

Adoptive Transfer↗
PubMed2003

Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes.

Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M. circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M. circinelloides a suitable organism for investigating some unresolved questions in RNA silencing.

Base Sequence↗
PubMed2002

Methods to estimate the proportion of plant and fungal RNA in an arbuscular mycorrhiza.

Arbuscular mycorrhizas are endosymbiotic associations formed between obligately biotrophic arbuscular mycorrhizal (AM) fungi and plant roots. The fungus and plant coexist in intimate contact as the fungus grows within the cortex of the root. RNA isolated from arbuscular mycorrhizas contains transcripts from both eukaryotic genomes. It is essential to be able to estimate the relative levels of fungal and plant RNA so that changes in plant and fungal gene expression can be evaluated during development of the AM symbiosis. Here we describe the design and use of specific plant and fungal internal transcribed spacer sequences and 18S rRNA probes to distinguish and quantify the relative levels of RNA of plant and fungal origin in samples from arbuscular mycorrhizas. We present two different methods. The first employs the most traditional method of transcript level analysis, namely northern blot analysis. The second one uses ribonuclease protection assays, which permit the analysis of transcript levels in a very small amount of tissue and are proving to be suitable for the analysis of gene expression in AM fungi. Analysis of tissues from a developing mycorrhiza showed that the relative levels of fungal RNA increased gradually as colonization of the root system progressed, reaching 5-12% in the most highly colonized samples.

Agaricales↗
PubMedDate not supplied

Prospects for dendritic cell vaccination against fungal infections in hematopoietic transplantation.

Dendritic cells (DCs) are uniquely able to initiate and control the immune response to fungi. DCs function at three levels in the manipulation of the immune response to these pathogens. First, they mount an immediate or innate response to them, for example, by producing inflammatory mediators upon capture and phagocytosis; second, through these preceding innate functions, they decode the fungus-associated information and translate it in qualitatively different Th responses, and third, they are key in containing and dampening inflammatory responses by tolerization through the induction of regulatory T cells (Treg). DCs sense fungi in a morphotype-specific manner, through the engagement of distinct recognition receptors ultimately affecting cytokine production and costimulation. Both myeloid and plasmacytoid murine and human DCs phagocytose fungi and undergo functional maturation in response to them. However, their activation program for cytokine production was different, being IL-12 mainly produced by myeloid DCs and IL-12, IL-10 and IFN-alpha mainly produced by plasmacytoid DCs. This resulted in a distinct ability for T cell priming, being Th1, Th2, and Treg differently activated by the different DC subsets. The ability of fungus-pulsed DCs to prime for Th1 and Th2 cell activation upon adoptive transfer in vivo correlated with the occurrence of resistance and susceptibility to the infections, respectively. Antifungal protective immunity was also induced upon adoptive transfer of DCs transfected with fungal RNA. The efficacy was restricted to DCs transfected with RNA from yeasts or conidia but not with RNA from fungal hyphae. The effect was fungus-specific, as no cross-protection was observed upon adoptive transfer of DCs pulsed with either fungal species. The infusion of fungus-pulsed or RNA-transfected DCs accelerated the recovery of functional antifungal Th1 responses in mice with allogeneic hematopoietic stem cell transplantation (HSCT) and affected the outcome of the infections. As the ability of phagocytose fungi was defective in peripheral DCs from patients with HSCT, soon after the transplant, our findings suggest that the adoptive transfer of DCs may restore immunocompetence in HSCT by contributing to the educational program of T cells. Thus, the remarkable functional plasticity of DCs in response to fungi can be exploited for the deliberate targeting of cells and pathways of cell-mediated immunity in response to fungal vaccines.

Adoptive Transfer↗
PubMed1985

Heterogeneity of 5S RNA in fungal ribosomes.

Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha). A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi. In addition, different 5S RNA's are present in Neurospora ribosomes. The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well.

Animals↗
PubMed2003

A dendritic cell vaccine against invasive aspergillosis in allogeneic hematopoietic transplantation.

Dendritic cells (DCs) have a remarkable functional plasticity in response to conidia and hyphae of the fungus Aspergillus fumigatus. In the present study we sought to assess the capacity of DCs activated by live fungi or fungal RNA to generate antifungal immunity in vivo. We found that both human and murine DCs pulsed with live fungi or transfected with fungal RNA underwent functional maturation, as revealed by the up-regulated expression of histocompatibility class II antigen and costimulatory molecules and the production of interleukin 12 (IL-12) in response to conidia or conidial RNA and of IL-4/IL-10 in response to hyphae or hyphal RNA. DCs pulsed with conidia or transfected with conidial RNA activated antigen-specific, interferon gamma (IFN-gamma)-producing T lymphocytes in vitro and in vivo on adoptive transfer in mice otherwise susceptible to aspergillosis. TH1-dependent antifungal resistance could also be induced in mice receiving allogeneic bone marrow transplants and was associated with an accelerated recovery of myeloid and lymphoid cells. Because the efficacy of the infusion of DCs was superior to that obtained on the adoptive transfer of Aspergillus-specific T cells, these results indicate the vaccinating potential of DCs pulsed with Aspergillus conidia or conidial RNA in hematopoietic transplantation.

Adoptive Transfer↗
PubMed2006

An efficient fungal RNA-silencing system using the DsRed reporter gene.

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.

Acremonium↗
PubMed2004

RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases.

The versatility of RNA-dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the kingdom Fungi. Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene-silencing processes, including heterochromatin regulation and RNA silencing in S. pombe and meiotic silencing and RNA silencing in N. crassa. Here we show that Aspergillus nidulans, another model fungus, does not require an RDRP for inverted repeat transgene (IRT)-induced RNA silencing. However, RDRP requirements may vary within the Aspergillus genus as genomic analysis indicates that A. nidulans, but not A. fumigatus or A. oryzae, has lost a QDE-1 ortholog, an RDRP associated with RNA silencing in N. crassa. We also provide evidence suggesting that 5' --> 3' transitive RNA silencing is not a significant aspect of A. nidulans IRT-RNA silencing. These results indicate a lack of conserved kingdom-wide requirements for RDRPs in fungal RNA silencing.

Aspergillus nidulans↗
PubMed2003

Differential expression of Glomus intraradices genes in external mycelium and mycorrhizal roots of tomato and barley.

Relative quantitative RT-PCR and western blotting were used to investigate the expression of three genes with potentially regulatory functions from the arbuscular mycorrhizal fungus Glomus intraradices in symbiosis with tomato and barley. Standardisation of total RNA per sample and determination of different ratios of plant and fungal RNA in roots as colonisation proceeded were achieved by relative quantitative RT-PCR using universal (NS1/NS21) and organism-specific rRNA primers. In addition, generic primers were designed for amplification of plant or fungal beta-tubulin genes and for plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as these have been suggested as useful controls in symbiotic systems. The fungal genes Ginmyc1 and Ginhb1 were expressed only in the external mycelium and not in colonised roots at both mRNA and protein levels, with the proteins detected almost exclusively in the insoluble fractions. In contrast, mRNA of Ginmyc2 was identified in both external and intraradical mycelium. In mycorrhizal roots, Ginmyc2 and fungal beta-tubulin mRNAs increased in proportion to fungal rRNA as colonisation proceeded, suggesting that accumulation reflected intraradical fungal growth. Fungal alpha-tubulin protein and beta-tubulin mRNA both appeared to be more abundantly accumulated in AM hyphae within heavily colonised roots than in external hyphae, relative to fungal rRNA. Tomato GAPDH mRNA accumulation was proportional to tomato rRNA, but accumulation of tomato beta-tubulin mRNA was reduced in colonised roots compared to non-mycorrhizal roots. These results provide novel evidence of differential spatial and temporal regulation of AM fungal genes, indicate that the expression of tubulin genes of both plant and fungus may be regulated during colonisation and validate the use of multiple 'control' genes in analysis of mycorrhizal gene expression.

Base Sequence↗
PubMed1988

Opposite effects of adenosine on two types of cAMP-induced gene expression in Dictyostelium indicate the involvement of at least two different intracellular pathways for the transduction of cAMP signals.

Adenosine promotes the cAMP-induced increase of mRNAs, probed with the cDNAs D11 and D14, which are preferentially expressed in prestalk cells, while it inhibits cAMP-induced prespore gene expression. Half-maximal inhibition of prespore gene expression occurs at about 300 muM, while prestalk stimulation by adenosine occurs at about 100-fold lower concentrations and requires the presence of cAMP. These results indicate that adenosine interferes with the transduction to cAMP to gene expression and suggest the involvement of two different adenosine target sites. Our data furthermore indicate that the transduction of extracellular cAMP to prespore gene or prestalk gene expression occurs via divergent pathways.

Adenosine↗
PubMed1990

cAMP regulation of the expression of prespore-specific genes, SP96 and Dp87, in disaggregated slug cells of Dictyostelium discoideum.

By Northern transfer and in vitro transcription assays, we examined how cAMP controlled the expression of prespore genes, Dp87 and SP96, in disaggregated slug cells of Dictyostelium discoideum. The transcription of these genes was halted soon after disaggregation and the remaining mRNAs were completely lost within 2 h. Addition of cAMP to cells which had lost the mRNAs induced the transcription of these genes, and the mRNAs were re-accumulated after a lag period of 30 min. The cAMP signal was transduced through the cell surface receptor. Protein synthesis was not needed for the induction of the transcription but was required for the accumulation of the mRNAs. We conclude that prespore gene expression is controlled by cAMP in two different ways: direct induction of transcription of the genes, and stabilization of the transcribed mRNAs by a protein(s) synthesized after addition of cAMP.

Blotting, Northern↗
PubMed1990

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.

Blotting, Northern↗
PubMed1998

Inhibition of RNA synthesis as a therapeutic strategy against Aspergillus and Fusarium: demonstration of in vitro synergy between rifabutin and amphotericin B.

We investigated the in vitro antifungal activity of amphotericin B, alone and in combination with rifabutin, an inhibitor of bacterial RNA polymerase, against 26 clinical isolates of Aspergillus and 25 clinical isolates of Fusarium. Synergy or additivism between these drugs was demonstrated against all isolates tested. Amphotericin B MICs were reduced upon combination with rifabutin from a mean of 0.65 microg/ml to a mean of 0.16 microg/ml against Aspergillus, and from a mean of 0.97 microg/ml to a mean of 0.39 microLg/ml against Fusarium (P < 0.000001 for both). Similarly, the MICs of rifabutin were reduced upon combination with amphotericin B from a mean of >32 microg/ml to a mean of 1.1 microg/ml against both fungi (P < 0.000001 for both). These positive interactions were corroborated by a colony count study with two Fusarium isolates, for which treatment with the combination of subinhibitory concentrations of amphotericin B (at concentrations 2- and 4-fold less than the MIC) and rifabutin (at concentrations ranging from 4- to 64-fold less than the MIC) resulted in 3.2-log reductions in colony counts compared to those after treatment with either drug alone. Inhibition of RNA synthesis was shown to be the mechanism of antifungal activity. These results suggest that inhibition of fungal RNA synthesis might be a potential target for antifungal therapy.

Amphotericin B↗