Ramón Lucas - Academia.edu (original) (raw)

Papers by Ramón Lucas

FEMS Microbiology Letters, 2001

The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACU... more The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni 2 affinity chromatography. The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting. For localisation studies A. nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA p , was derived. Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Archives of Microbiology, 1994

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidu... more The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucoseinduced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.

Fungal Genetics and Biology, 2008

Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifu... more Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (DacuD strains) or malate synthase (DacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of DacuD and DacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the DacuD and DacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.

Archives of Microbiology, 2000

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regul... more In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

Fungal Genetics and …, 2003

Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocom... more Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocompromised patients. Functional genomics in this fungus will aid the discovery of novel antifungal drugs to treat invasive aspergillosis. However, there is still a need for appropriate molecular ...

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty a... more Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid b-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DacuH strain on acetate and longchain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

Archives of …, 1997

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a s... more Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 x 10(7) viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole-) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole- mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim- (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 x g supernatant, but produced wild-type levels of penicillin.

Archives of Microbiology

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies... more In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolas...

FEMS Microbiology Letters, 2001

The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACU... more The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni 2 affinity chromatography. The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting. For localisation studies A. nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA p , was derived. Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

Archives of Microbiology, 1994

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidu... more The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucoseinduced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.

Fungal Genetics and Biology, 2008

Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifu... more Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (DacuD strains) or malate synthase (DacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of DacuD and DacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the DacuD and DacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.

Archives of Microbiology, 2000

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regul... more In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

Fungal Genetics and …, 2003

Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocom... more Aspergillus fumigatus causes invasive aspergillosis, a mycosis that is usually fatal in immunocompromised patients. Functional genomics in this fungus will aid the discovery of novel antifungal drugs to treat invasive aspergillosis. However, there is still a need for appropriate molecular ...

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty a... more Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid b-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DacuH strain on acetate and longchain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

Archives of …, 1997

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a s... more Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 x 10(7) viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole-) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole- mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim- (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 x g supernatant, but produced wild-type levels of penicillin.

Archives of Microbiology

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies... more In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolas...