Final Report Summary - CELLWALLPATH (Identification of new molecules able to bypass the cell wall compensatory pathways in the pathogenic fungus Aspergillus fumigatus
Main scientific goals achieved during the project
Scientific relevance of the project
A. fumigatus is the most important airborne human-pathogenic fungus. It has the potential to cause life-threatening diseases in immunocompromised patients named as aspergillosis. In the last two decades, the incidence of invasive aspergillosis has tremendously increased since therapeutic and diagnostic interventions are still limited (Brakhage 2005, J. Curr. Drug Targets 6:875).
Clinical research identified the fungal cell wall as a promising target to cure pathogenic mould. The cell wall of A. fumigatus contains a unique polysaccharidic composition that does not exist for human cells. This barrier enables the fungus to resist against external aggressions, but, at the same time, it is its Achilles' heel since it is a major drug target as shown by the commercial launch of Echinocandins that block cell wall biosynthesis. Environmental changes are sensed by A. fumigatus and transduced via signal transduction pathways that lead to the activation of response strategies. Comparative studies highlighted that MAPKs, calmodulin / calcineurin, TOR and Ras / cAMP signalling pathways are well conserved in eukaryotic organisms (Wullschleger et al. 2006, Cell, 124:471; Rispail et al. 2009, Fungal Genet. Biol. 46:287). Computer analysis of the whole genome of A. fumigatus and comparison with genes known to be involved in fungal signal transduction pathways led to the identification of many putative elements in this fungus. The analysis of the A. fumigatus genome revealed the presence of four genes that encode MAPKs. One of those, named mpkA, was shown to be required for cell wall integrity but no influence on virulence of the mpkA deletion mutant was observed in a murine infection model (Valiante et al. 2008, Fungal Genet. Biol. 45:618).
Signal transduction pathways can act separately but evidence of interaction between pathways is emerging (Bernejo et al. 2008, Mol. Biol. Cell 19:1113).
Identification of cell wall integrity compensatory pathways in A. fumigatus
A physical and chemical defence used by fungi to protect themselves against external stress is the production of melanin. It was shown that A. fumigatus produces two types of melanin: the 1,8-dihydroxynaphthalene (DHN) melanin, derived from activated acetate, and an alternative type of melanin, designated pyomelanin, via tyrosine degradation. Pyomelanin is formed during the degradation of L-tyrosine. The intermediate 4-hydroxyphenylpyruvate is converted by the enzyme 4-hydroxyphenylpyruvate dioxygenase (HppD) to homogentisic acid (HGA). HGA can either be degraded further to 4-maleylacetoacetate by the homogentisate dioxygenase (HmgA) or can polymerise to pyomelanin. We already showed that cell wall stress is involved in the regulation of pyomelanin formation via the tyrosine degradation pathway in A. fumigatus (Valiante et al., 2009, Fungal Genet. Biol. 46:909).
DHN melanin is synthesised by enzymes whose corresponding genes are organised in a cluster. The key enzyme of this pathway represents a polyketide synthase encoded by the pksP gene (also known as alb1). The pksP mutant exhibits white conidia, sensitivity to reactive oxygen species (ROS) and reduced virulence. So far, it was shown that the regulation of the pksP gene cluster was cAMP-dependent. Growing MAPKs mutants in liquid cultures, we observed that the mpkB and mpkC mutant strains release a dark pigment in the media. Northern blot analysis showed that the pksP gene resulted to be constitutively expressed in these two mutant strains. Construction of the mpkB / pksP and the mpkC / pksP double mutant strains confirmed that the released pigment is DHN melanin. These results suggested that the putative pheromone pathway and the MpkC pathway negatively influence the melanin production in A. fumigatus. Even more, deletion of the mpkA gene in the mpkB mutant strain repressed this phenotype. As a conclusion, we observed that the pksP gene cluster is most likely regulated by all MAP kinase signalling pathways present in A. fumigatus, and it could potentially be used as marker to study cross-talk interaction among the MAPK signalling pathways.
Identification of compensatory pathways in response to antifungal drugs
In order to identify response genes involved in Caspofungin (CAS) response, we performed a transcriptome analysis using Ribonucleic acid (RNA)-seq approach, on A. fumigatus. We performed this analysis including two wild-type strains. The obtained results confirmed that CAS effects cellular and carbohydrate metabolism as a consequence of cell wall stress. Moreover, data demonstrated that additionally to cell wall stress, CAS affects cell membrane inducing osmolarity stress. The increase number of genes involved in transport and oxi-reduction processes enriched during the analysis proved this.
In order to identify pathways involved in the CAS stress response, we included in the analysis also the mpkA and the sakA mutant strains. Analysis on the mpkA mutant strain revealed that many responsive genes involved in cell wall stress responses resulted to be not induced. This proved the central role played by the cell wall integrity pathway during CAS response. On the other hand, genes involved in osmolarity response, and in the transport and oxi-reduction processes, resulted to be not induced in the sakA mutant strain. With this experiment we demonstrated that the response during CAS stress involves different signalling, that can cope for compensatory effects during CAS response. With this study we want to better identify CAS targets, in order to clarify the pharmacodinamic of this important antifungal drug, with the aim of using this information to increase the activity of this drug.
Screening of natural product libraries to discover new antifungal combinations
The vertiginous increase of invasive fungal infections, the emergency of antifungal drugs resistance and the lack of new antifungals introduced in the clinic, have led the scientific community to search for new strategies to combat these life-threatening diseases. The discovery of novel compounds displaying synergism with antifungals currently available appears to be the most reasonable strategy. CAS, an antifungal with no target in mammalian cells, is an excellent candidate to be used in combination therapy since it presents a few pharmacokinetic drug interactions. We identified a new peptide, named as MDN-0010, able to potentiate 4.5 times the CAS effect against A. fumigatus. To investigate the synergistic effect of CAS and MDN-0010 compound, different transcriptome analyses were performed using deep RNA-seq approach. These studies allowed the identification of the genes differentially regulated in response to drug stresses, and results obtained suggest that MDN-0010 acts by reducing the A. fumigatus response against CAS getting less specific and increasing the toxic effect. MDN-0010 was also tested using the A. fumigatus mutant collection. Additionally, a sensitive cytotoxicity assay was performed using the normal human hepatocyte cell line (Fa2N-4).
Generation of recombinant A. fumigatus strains
How compensatory reactions are activated is still unclear; in particular the cross-talk interaction occurring among the signal transduction cascades has not been elucidated yet. The role of protein kinases in signalling pathways was well established. These proteins are undoubtedly the 'positive' activators of the transduction machinery that act in the cells. Nevertheless, phosphorylation balance plays an important role in the compensatory mechanisms among different signalling pathways. Moreover, protein phosphatases, and their cognate kinases, are relevant to provide both spatial and temporal regulation during cell processes. Aim of this project was to study the role of protein phosphatases in A. fumigatus. Automated ontology classification identified 24 putative protein phosphatases in A. fumigatus (Wolstencroft et al. 2006, Bioinformatics, 22:e530).
A. fumigatus genome was released in 2005 (Nierman et al. 2005, Nature, 438:1151). The gene annotation was improved in the last year, but the classification was always some time superficial. The first step was to confirm the number of total phosphatases present in the A. fumigatus genome. Comparative blastp and tblastn analysis identified 33 putative protein phosphatases: 5 tyrosine-specific phosphatases (including one low molecular weight phosphatase), 18 serine/threonine specific phosphatases, 7 dual specificity phosphatases, 1 specific myotubularin, 1 CDC25, and 1 CDT posphatase.
We established a fast cloning methodology based on the gene Gateway system exploiting Saccharomyces cerevisiae as a tool (Colot et al. 2006, PNAS 103:10352). For this purpose, we used the Escherichia coli hph gene, coding for the hygromycin B resistant gene, as dominant marker. The hph gene was already cloned under the control of the constitutive gpdA promoter, suitable for expression in S. cerevisiae and A. fumigatus. Flanking regions for each gene and the gpdA-hph cassette were amplified by Polymerase chain reaction (PCR), pooled with a pre-digested 2 m plasmid and used to transform S. cerevisiae. The obtained plasmids were screened by PCR and used for transformation. This approach was successfully used to delete 28 putative protein phosphatases genes and to create 5 inducible mutant strains for the genes that resulted to be essential.
The screening of the phosphatase library revealed that many genes are difficult to characterise, and most likely there is redundancy among the different phosphatases. However, phonotypical analysis revealed that at list two different serine / threonine protein phosphatases are important for cell wall maintaining. Further analyses are ongoing.
Project management
Benefit to the career of the researcher from the period of re-integration
To be part of the Marie Curie program had great benefits to my career. I have been involved in the study of signalling pathways in A. fumigatus. The studies on cell signalling pathways have a primary importance for infection biology. In the last three years I specialised myself in computational biology in order to lead research groups in system biology. I supervised different students (2 PhDs, 3 Master students, 2 Bachelor students) and I am currently leading the 'Molecular Biotechnology' unit in the 'Molecular and Applied Microbiology' department. 1 post doc, 2 PhD students and one technician compose my group.
I started cooperation with different national and international institutes including private companies. My contract is going to expire at the end of this year, but I have been granted for the next four year. My actual position is not stable, but I am quite optimist).
Scientific relevance of the project
A. fumigatus is the most important airborne human-pathogenic fungus. It has the potential to cause life-threatening diseases in immunocompromised patients named as aspergillosis. In the last two decades, the incidence of invasive aspergillosis has tremendously increased since therapeutic and diagnostic interventions are still limited (Brakhage 2005, J. Curr. Drug Targets 6:875).
Clinical research identified the fungal cell wall as a promising target to cure pathogenic mould. The cell wall of A. fumigatus contains a unique polysaccharidic composition that does not exist for human cells. This barrier enables the fungus to resist against external aggressions, but, at the same time, it is its Achilles' heel since it is a major drug target as shown by the commercial launch of Echinocandins that block cell wall biosynthesis. Environmental changes are sensed by A. fumigatus and transduced via signal transduction pathways that lead to the activation of response strategies. Comparative studies highlighted that MAPKs, calmodulin / calcineurin, TOR and Ras / cAMP signalling pathways are well conserved in eukaryotic organisms (Wullschleger et al. 2006, Cell, 124:471; Rispail et al. 2009, Fungal Genet. Biol. 46:287). Computer analysis of the whole genome of A. fumigatus and comparison with genes known to be involved in fungal signal transduction pathways led to the identification of many putative elements in this fungus. The analysis of the A. fumigatus genome revealed the presence of four genes that encode MAPKs. One of those, named mpkA, was shown to be required for cell wall integrity but no influence on virulence of the mpkA deletion mutant was observed in a murine infection model (Valiante et al. 2008, Fungal Genet. Biol. 45:618).
Signal transduction pathways can act separately but evidence of interaction between pathways is emerging (Bernejo et al. 2008, Mol. Biol. Cell 19:1113).
Identification of cell wall integrity compensatory pathways in A. fumigatus
A physical and chemical defence used by fungi to protect themselves against external stress is the production of melanin. It was shown that A. fumigatus produces two types of melanin: the 1,8-dihydroxynaphthalene (DHN) melanin, derived from activated acetate, and an alternative type of melanin, designated pyomelanin, via tyrosine degradation. Pyomelanin is formed during the degradation of L-tyrosine. The intermediate 4-hydroxyphenylpyruvate is converted by the enzyme 4-hydroxyphenylpyruvate dioxygenase (HppD) to homogentisic acid (HGA). HGA can either be degraded further to 4-maleylacetoacetate by the homogentisate dioxygenase (HmgA) or can polymerise to pyomelanin. We already showed that cell wall stress is involved in the regulation of pyomelanin formation via the tyrosine degradation pathway in A. fumigatus (Valiante et al., 2009, Fungal Genet. Biol. 46:909).
DHN melanin is synthesised by enzymes whose corresponding genes are organised in a cluster. The key enzyme of this pathway represents a polyketide synthase encoded by the pksP gene (also known as alb1). The pksP mutant exhibits white conidia, sensitivity to reactive oxygen species (ROS) and reduced virulence. So far, it was shown that the regulation of the pksP gene cluster was cAMP-dependent. Growing MAPKs mutants in liquid cultures, we observed that the mpkB and mpkC mutant strains release a dark pigment in the media. Northern blot analysis showed that the pksP gene resulted to be constitutively expressed in these two mutant strains. Construction of the mpkB / pksP and the mpkC / pksP double mutant strains confirmed that the released pigment is DHN melanin. These results suggested that the putative pheromone pathway and the MpkC pathway negatively influence the melanin production in A. fumigatus. Even more, deletion of the mpkA gene in the mpkB mutant strain repressed this phenotype. As a conclusion, we observed that the pksP gene cluster is most likely regulated by all MAP kinase signalling pathways present in A. fumigatus, and it could potentially be used as marker to study cross-talk interaction among the MAPK signalling pathways.
Identification of compensatory pathways in response to antifungal drugs
In order to identify response genes involved in Caspofungin (CAS) response, we performed a transcriptome analysis using Ribonucleic acid (RNA)-seq approach, on A. fumigatus. We performed this analysis including two wild-type strains. The obtained results confirmed that CAS effects cellular and carbohydrate metabolism as a consequence of cell wall stress. Moreover, data demonstrated that additionally to cell wall stress, CAS affects cell membrane inducing osmolarity stress. The increase number of genes involved in transport and oxi-reduction processes enriched during the analysis proved this.
In order to identify pathways involved in the CAS stress response, we included in the analysis also the mpkA and the sakA mutant strains. Analysis on the mpkA mutant strain revealed that many responsive genes involved in cell wall stress responses resulted to be not induced. This proved the central role played by the cell wall integrity pathway during CAS response. On the other hand, genes involved in osmolarity response, and in the transport and oxi-reduction processes, resulted to be not induced in the sakA mutant strain. With this experiment we demonstrated that the response during CAS stress involves different signalling, that can cope for compensatory effects during CAS response. With this study we want to better identify CAS targets, in order to clarify the pharmacodinamic of this important antifungal drug, with the aim of using this information to increase the activity of this drug.
Screening of natural product libraries to discover new antifungal combinations
The vertiginous increase of invasive fungal infections, the emergency of antifungal drugs resistance and the lack of new antifungals introduced in the clinic, have led the scientific community to search for new strategies to combat these life-threatening diseases. The discovery of novel compounds displaying synergism with antifungals currently available appears to be the most reasonable strategy. CAS, an antifungal with no target in mammalian cells, is an excellent candidate to be used in combination therapy since it presents a few pharmacokinetic drug interactions. We identified a new peptide, named as MDN-0010, able to potentiate 4.5 times the CAS effect against A. fumigatus. To investigate the synergistic effect of CAS and MDN-0010 compound, different transcriptome analyses were performed using deep RNA-seq approach. These studies allowed the identification of the genes differentially regulated in response to drug stresses, and results obtained suggest that MDN-0010 acts by reducing the A. fumigatus response against CAS getting less specific and increasing the toxic effect. MDN-0010 was also tested using the A. fumigatus mutant collection. Additionally, a sensitive cytotoxicity assay was performed using the normal human hepatocyte cell line (Fa2N-4).
Generation of recombinant A. fumigatus strains
How compensatory reactions are activated is still unclear; in particular the cross-talk interaction occurring among the signal transduction cascades has not been elucidated yet. The role of protein kinases in signalling pathways was well established. These proteins are undoubtedly the 'positive' activators of the transduction machinery that act in the cells. Nevertheless, phosphorylation balance plays an important role in the compensatory mechanisms among different signalling pathways. Moreover, protein phosphatases, and their cognate kinases, are relevant to provide both spatial and temporal regulation during cell processes. Aim of this project was to study the role of protein phosphatases in A. fumigatus. Automated ontology classification identified 24 putative protein phosphatases in A. fumigatus (Wolstencroft et al. 2006, Bioinformatics, 22:e530).
A. fumigatus genome was released in 2005 (Nierman et al. 2005, Nature, 438:1151). The gene annotation was improved in the last year, but the classification was always some time superficial. The first step was to confirm the number of total phosphatases present in the A. fumigatus genome. Comparative blastp and tblastn analysis identified 33 putative protein phosphatases: 5 tyrosine-specific phosphatases (including one low molecular weight phosphatase), 18 serine/threonine specific phosphatases, 7 dual specificity phosphatases, 1 specific myotubularin, 1 CDC25, and 1 CDT posphatase.
We established a fast cloning methodology based on the gene Gateway system exploiting Saccharomyces cerevisiae as a tool (Colot et al. 2006, PNAS 103:10352). For this purpose, we used the Escherichia coli hph gene, coding for the hygromycin B resistant gene, as dominant marker. The hph gene was already cloned under the control of the constitutive gpdA promoter, suitable for expression in S. cerevisiae and A. fumigatus. Flanking regions for each gene and the gpdA-hph cassette were amplified by Polymerase chain reaction (PCR), pooled with a pre-digested 2 m plasmid and used to transform S. cerevisiae. The obtained plasmids were screened by PCR and used for transformation. This approach was successfully used to delete 28 putative protein phosphatases genes and to create 5 inducible mutant strains for the genes that resulted to be essential.
The screening of the phosphatase library revealed that many genes are difficult to characterise, and most likely there is redundancy among the different phosphatases. However, phonotypical analysis revealed that at list two different serine / threonine protein phosphatases are important for cell wall maintaining. Further analyses are ongoing.
Project management
Benefit to the career of the researcher from the period of re-integration
To be part of the Marie Curie program had great benefits to my career. I have been involved in the study of signalling pathways in A. fumigatus. The studies on cell signalling pathways have a primary importance for infection biology. In the last three years I specialised myself in computational biology in order to lead research groups in system biology. I supervised different students (2 PhDs, 3 Master students, 2 Bachelor students) and I am currently leading the 'Molecular Biotechnology' unit in the 'Molecular and Applied Microbiology' department. 1 post doc, 2 PhD students and one technician compose my group.
I started cooperation with different national and international institutes including private companies. My contract is going to expire at the end of this year, but I have been granted for the next four year. My actual position is not stable, but I am quite optimist).