RESUMEN
The fungal wheat pathogen Zymoseptoria tritici causes major crop losses as the causal agent of the disease Septoria tritici blotch. The infection cycle of Z. tritici displays two distinct phases, beginning with an extended symptomless phase of 1-2 weeks, before the fungus induces host cell death and tissue collapse in the leaf. Recent evidence suggests that the fungus uses little host-derived nutrition during asymptomatic colonisation, raising questions as to the sources of energy required for this initial growth phase. Autophagy is crucial for the pathogenicity of other fungal plant pathogens through its roles in supporting cellular differentiation and growth under starvation. Here we characterised the contributions of the autophagy genes ZtATG1 and ZtATG8 to the development and virulence of Z. tritici. Deletion of ZtATG1 led to inhibition of autophagy but had no impact on starvation-induced hyphal differentiation or virulence, suggesting that autophagy is not required for Z. tritici pathogenicity. Contrastingly, ZtATG8 deletion delayed the transition to necrotrophic growth, despite having no influence on filamentous growth under starvation, pointing to an autophagy-independent role of ZtATG8 during Z. tritici infection. To our knowledge, this study represents the first to find autophagy not to contribute to the virulence of a fungal plant pathogen, and reveals novel roles for different autophagy-associated proteins in Z. tritici.
Asunto(s)
Ascomicetos , Enfermedades de las Plantas , Virulencia/genética , Enfermedades de las Plantas/microbiología , Autofagia/genéticaRESUMEN
Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections.
Asunto(s)
Antifúngicos/farmacología , Candidiasis/tratamiento farmacológico , Farmacorresistencia Fúngica/genética , Regulación Fúngica de la Expresión Génica , Antifúngicos/uso terapéutico , Candida glabrata/genética , Candidiasis/microbiología , Fluconazol/farmacología , Fluconazol/uso terapéutico , Mutación con Ganancia de Función , Histona Acetiltransferasas/genética , Humanos , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Mutaciones Letales SintéticasRESUMEN
Libraries of protein-encoding sequences can be generated by identification of open reading frames (ORFs) from a genome of choice that are then assembled into collections of plasmids termed ORFeome libraries. These represent powerful resources to facilitate functional genomic characterization of genes and their encoded products. Here, we report the generation of an ORFeome for Zymoseptoria tritici, which causes the most serious disease of wheat in temperate regions of the world. We screened the genome of strain IP0323 for high confidence gene models, identifying 4,075 candidates from 10,933 predicted genes. These were amplified from genomic DNA, were cloned into the Gateway entry vector pDONR207, and were sequenced, providing a total of 3,022 quality-controlled plasmids. The ORFeome includes genes predicted to encode effectors (n = 410) and secondary metabolite biosynthetic proteins (n = 171) in addition to genes residing at dispensable chromosomes (n = 122) or those that are preferentially expressed during plant infection (n = 527). The ORFeome plasmid library is compatible with our previously developed suite of Gateway destination vectors, which have various combinations of promoters, selection markers, and epitope tags. The Z. tritici ORFeome constitutes a powerful resource for functional genomics and offers unparalleled opportunities to understand the biology of Z. tritici.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Asunto(s)
Ascomicetos , Genoma Fúngico , Biblioteca Genómica , Genómica , Sistemas de Lectura Abierta , Ascomicetos/genética , Genoma Fúngico/genética , Genómica/métodos , Sistemas de Lectura Abierta/genética , Triticum/microbiologíaRESUMEN
Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.
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Biopelículas/crecimiento & desarrollo , Biomarcadores/análisis , Candida/clasificación , Candida/genética , Candidiasis/genética , Proteínas Fúngicas/genética , Candida/crecimiento & desarrollo , Candidiasis/microbiología , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN de Hongos/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Especificidad de la EspecieRESUMEN
The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida glabrata/genética , Farmacorresistencia Fúngica/genética , Equinocandinas/farmacología , Biopelículas/crecimiento & desarrollo , Candida glabrata/crecimiento & desarrollo , Candidiasis/tratamiento farmacológico , Caspofungina , Pared Celular/efectos de los fármacos , Pared Celular/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Técnicas de Inactivación de Genes , Biblioteca de Genes , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Presión Osmótica , FenotipoRESUMEN
Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a ß-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.
Asunto(s)
Fusión Artificial Génica , Ascomicetos/genética , Expresión Génica , Nitrato-Reductasa/genética , Regiones Promotoras Genéticas , Ascomicetos/crecimiento & desarrollo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Nitratos/metabolismo , Recombinación GenéticaRESUMEN
The trend for large-scale genetic and phenotypic screens has revealed a wealth of information on biological systems. A major challenge is understanding how genes function and putative roles in networks. The majority of current gene knowledge is garnered from studies utilising the model yeast Saccharomyces cerevisiae. We demonstrate that synthetic dosage lethal genetic array methodologies can be used to study genetic networks in other yeasts, namely the fungal pathogen Candida glabrata, which has limited forward genetic tools, due to the lack of 'natural' mating. We performed two SDL screens in S. cerevisiae, overexpressing the transcriptional regulator UME6 as bait in the first screen and its C. glabrata ortholog CAGL0F05357g in the second. Analysis revealed that SDL maps share 204 common interactors, with 10 genetic interactions unique to C. glabrata indicating a level of genetic rewiring, indicative of linking genotype to phenotype in fungal pathogens. This was further validated by incorporating our results into the global genetic landscape map of the cell from Costanzo et al. to identify common and novel gene attributes. This data demonstrated the utility large data sets and more robust analysis made possible by interrogating exogenous genes in the context of the eukaryotic global genetic landscape.
Asunto(s)
Candida glabrata/genética , Redes Reguladoras de Genes , Pruebas Genéticas/métodos , Saccharomyces cerevisiae/genética , Cruzamientos Genéticos , Análisis Mutacional de ADN , Viabilidad Microbiana , Saccharomyces cerevisiae/fisiologíaRESUMEN
In recent years, there has been a noticeable rise in fungal infections related to non-albicans Candida species, including Candida glabrata which has both intrinsic resistance to and commonly acquired resistance to azole antifungals. Phylogenetically, C. glabrata is more closely related to the mostly non-pathogenic model organism Saccharomyces cerevisiae than to other Candida species. Despite C. glabrata's designation as a pathogen by Wickham in 1957, relatively little is known about its mechanism of virulence. Over the past few years, technology to analyse the molecular basis of infection has developed rapidly, and here we briefly review the major advances in tools and technologies available to explore and investigate the virulence of C. glabrata that have occurred over the past decade.
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Candida glabrata/crecimiento & desarrollo , Candida glabrata/genética , Genética Microbiana/métodos , Biología Molecular/métodos , Factores de Virulencia/genética , Animales , Candidiasis/microbiología , Candidiasis/patología , Modelos Animales de Enfermedad , Genética Microbiana/tendencias , Humanos , Biología Molecular/tendencias , VirulenciaRESUMEN
The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.
Asunto(s)
Candida glabrata/genética , Proteínas Fúngicas/genética , Manosiltransferasas/genética , Mutación , Animales , Candida glabrata/enzimología , Candida glabrata/patogenicidad , Candidiasis/microbiología , Secuencia de Carbohidratos , Línea Celular , Pared Celular/genética , Pared Celular/metabolismo , Células Endoteliales/citología , Células Endoteliales/microbiología , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Glicosilación , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estimación de Kaplan-Meier , Espectroscopía de Resonancia Magnética , Masculino , Mananos/química , Mananos/metabolismo , Manosiltransferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Virulencia/genéticaRESUMEN
Yeast cells are able to tolerate and adapt to a variety of environmental stresses. An essential aspect of stress adaptation is the regulation of monovalent ion concentrations. Ion regulation determines many fundamental physiological parameters, such as cell volume, membrane potential, and intracellular pH. It is achieved through the concerted activities of multiple cellular components, including ion transporters and signaling molecules, on both short and long time scales. Although each component has been studied in detail previously, it remains unclear how the physiological parameters are maintained and regulated by the concerted action of all components under a diverse range of stress conditions. In this study, we have constructed an integrated mathematical model of ion regulation in Saccharomyces cerevisiae to understand this coordinated adaptation process. Using this model, we first predict that the interaction between phosphorylated Hog1p and Tok1p at the plasma membrane inhibits Tok1p activity and consequently reduces Na(+) influx under NaCl stress. We further characterize the impacts of NaCl, sorbitol, KCl and alkaline pH stresses on the cellular physiology and the differences between the cellular responses to these stresses. We predict that the calcineurin pathway is essential for maintaining a non-toxic level of intracellular Na(+) in the long-term adaptation to NaCl stress, but that its activation is not required for maintaining a low level of Na(+) under other stresses investigated. We provide evidence that, in addition to extrusion of toxic ions, Ena1p plays an important role, in some cases alongside Nha1p, in re-establishing membrane potential after stress perturbation. To conclude, this model serves as a powerful tool for both understanding the complex system-level properties of the highly coordinated adaptation process and generating further hypotheses for experimental investigation.
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Iones/metabolismo , Modelos Teóricos , Saccharomyces cerevisiae/metabolismo , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Fosforilación , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Cloruro de Sodio/metabolismo , Estrés FisiológicoRESUMEN
Understanding how populations and communities respond to competition is a central concern of ecology. A seminal theoretical solution first formalised by Levins (and re-derived in multiple fields) showed that, in theory, the form of a trade-off should determine the outcome of competition. While this has become a central postulate in ecology it has evaded experimental verification, not least because of substantial technical obstacles. We here solve the experimental problems by employing synthetic ecology. We engineer strains of Escherichia coli with fixed resource allocations enabling accurate measurement of trade-off shapes between bacterial survival and multiplication in multiple environments. A mathematical chemostat model predicts different, and experimentally verified, trajectories of gene frequency changes as a function of condition-specific trade-offs. The results support Levins' postulate and demonstrates that otherwise paradoxical alternative outcomes witnessed in subtly different conditions are predictable.
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Ecosistema , Escherichia coli/fisiología , Modelos Biológicos , Proteínas Bacterianas/metabolismo , Escherichia coli/crecimiento & desarrollo , Viabilidad Microbiana , Factor sigma/metabolismoRESUMEN
Alkaline pH adaptation represents an important environmental stress response in Aspergillus nidulans. It is mediated by the pal signalling pathway and the PacC transcription factor. Although studied extensively experimentally, the activation mechanism of PacC has not been quantified, and it is not clear how this activation is regulated. Here, by constructing mathematical models, we first show that the pattern of PacC activation observed in previously published experiments cannot be explained based on existing knowledge about PacC activation. Extending the model with a negative feedback loop is necessary to produce simulation results that are consistent with the data, suggesting the existence of a negative feedback loop in the PacC activation process. This extended model is then validated against published measurements for cells with drug treatment and mutant cells. Furthermore, we investigate the role of an intermediate form of PacC in the PacC activation process, and propose experiments that can be used to test our predictions. Our work illustrates how mathematical models can be used to uncover regulatory mechanisms in the transcription regulation, and generate hypotheses that guide further laboratory investigations.
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Aspergillus nidulans/genética , Retroalimentación Fisiológica/fisiología , Proteínas Fúngicas/agonistas , Modelos Biológicos , Factores de Transcripción/agonistas , Activación Transcripcional , Simulación por Computador , Regulación Fúngica de la Expresión Génica , Concentración de Iones de Hidrógeno , Estrés Fisiológico/genética , Estrés Fisiológico/fisiologíaRESUMEN
BACKGROUND: In biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. SCOPE OF THE REVIEW: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. MAJOR CONCLUSIONS: Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. GENERAL SIGNIFICANCE: Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms.
Asunto(s)
Respiración de la Célula/fisiología , Saccharomyces cerevisiae/metabolismo , Respiración de la Célula/genética , Modelos Biológicos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.
Asunto(s)
Candida albicans/fisiología , Candida glabrata/fisiología , Viabilidad Microbiana/efectos de los fármacos , Estrés Fisiológico , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Candida glabrata/efectos de los fármacos , Candida glabrata/crecimiento & desarrollo , Medios de Cultivo/química , Humanos , Micología/métodos , Compuestos Nitrosos/toxicidad , Presión Osmótica , Estrés Oxidativo , TemperaturaRESUMEN
The osmotic stress response signalling pathway of the model yeast Saccharomyces cerevisae is crucial for the survival of cells under osmotic stress, and is preserved to varying degrees in other related fungal species. We apply a method for inference of ancestral states of characteristics over a phylogeny to 17 fungal species to infer the maximum likelihood estimate of presence or absence in ancestral genomes of genes involved in osmotic stress response. The same method allows us furthermore to perform a statistical test for correlated evolution between genes. Where such correlations exist within the osmotic stress response pathway of S. cerevisae, we have used this in order to predict and subsequently test for the presence of physical protein-protein interactions in an attempt to detect novel interactions. Finally we assess the relevance of observed evolutionary correlations in predicting protein interactions in light of the experimental results. We do find that correlated evolution provides some useful information for the prediction of protein-protein interactions, but that these alone are not sufficient to explain detectable patterns of correlated evolution.
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Evolución Biológica , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Levaduras/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Ósmosis , Filogenia , Unión Proteica , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Levaduras/clasificación , Levaduras/metabolismoRESUMEN
Candida glabrata is a major fungal pathogen of humans, and the virulence of C. glabrata is increased by inactivation of the transcription factor, Ace2. Our previous examination of the effects of Ace2 inactivation upon the intracellular proteome suggested that the hypervirulence of C. glabrata ace2 mutants might be caused by differences in the secretome. Therefore in this study we have characterised the C. glabrata secretome and examined the effects of Ace2 inactivation upon this extracellular proteome. We have identified 31 distinct proteins in the secretome of wild-type C. glabrata cells by MS/MS of proteins that were precipitated from the growth medium and enriched by affinity chromatography on concanavalin A. Most of these proteins are predicted to be cell wall proteins, cell wall modifying enzymes and aspartyl proteinases. The endochitinase Cts1 and the endoglucanase Egt2 were not detected in the C. glabrata secretome following Ace2 inactivation. This can account for the cell separation defect of C. glabrata ace2 cells. Ace2 inactivation also resulted in the detection of new proteins in the C. glabrata secretome. The release of such proteins might contribute to the hypervirulence of ace2 cells.
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Candida glabrata/química , Proteínas Fúngicas/metabolismo , Proteoma/análisis , Candida glabrata/efectos de los fármacos , Candida glabrata/metabolismo , Candida glabrata/patogenicidad , Pared Celular/química , Doxiciclina/farmacología , Espacio Extracelular/química , Proteínas Fúngicas/genética , Glicosilación , Señales de Clasificación de Proteína , Proteoma/química , Transcripción Genética , VirulenciaRESUMEN
The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus L-ornithine-N5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog-and the fungal siderophore system in general-in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.
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Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Proteínas Fúngicas/genética , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Fenotipo , Sideróforos/biosíntesis , Animales , Aspergillus fumigatus/metabolismo , Secuencia de Bases , Northern Blotting , Cromatografía Líquida de Alta Presión , Cartilla de ADN , ADN Complementario/genética , Ratones , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutación/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.
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Adaptación Fisiológica/genética , Aspergilosis , Aspergillus fumigatus/genética , Regulación Viral de la Expresión Génica/fisiología , Animales , Aspergillus fumigatus/patogenicidad , Perfilación de la Expresión Génica , Genes Virales/fisiología , Ratones , Telómero , Virulencia/genéticaRESUMEN
BACKGROUND: Invasive aspergillosis (IA) is the most common cause of death associated with fungal infection in the developed world. Historically, susceptibility to IA has been associated with prolonged neutropenia; however, IA has now become a major problem in patients on calcineurin inhibitors and allogenic hematopoietic stem cell transplant patients following engraftment. These observations suggest complex cellular mechanisms govern immunity to IA. METHODS: To characterize the key early events that govern outcome from infection with Aspergillus fumigatus, we performed a comparative immunochip microarray analysis of the pulmonary transcriptional response to IA between cyclophosphamide-treated mice and immunocompetent mice at 24 h after infection. RESULTS: We demonstrate that death due to infection is associated with a failure to generate an incremental interferon-gamma response, increased levels of interleukin-5 and interleukin-17a transcript, coordinated expression of a network of tumor necrosis factor-alpha-related genes, and increased levels of tumor necrosis factor-alpha. In contrast, clearance of infection is associated with increased expression of a number genes encoding proteins involved in innate pathogen clearance, as well as apoptosis and control of inflammation. CONCLUSION: This first organ-level immune response transcriptional analysis for IA has enabled us to gain new insights into the mechanisms that govern fungal immunity in the lung.