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RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

O'Keeffe G, Hammel S, Owens RA, Keane TM, Fitzpatrick DA, Jones GW, Doyle S - BMC Genomics (2014)

Bottom Line: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated.Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin.Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, National University of Ireland Maynooth, Maynooth, Co, Kildare, Ireland. sean.doyle@nuim.ie.

ABSTRACT

Background: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin.

Results: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin.

Conclusions: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.

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Levels of fumagillin, pseurotin A, fumitremorgin C, tryprostatin B and tryprostatin A inA. fumigatuswild-type and ∆gliTfollowing 96 h growth under secondary metabolite inducing conditions. Fumagillin (A) and pseurotin A (B) production was significantly reduced in A. fumigatus ∆gliT compared to wild-type (*p =0.0471 and * p =0.0297, respectively). There was no significant difference in fumitremorgin C (C) levels, while brevianamide F (D) levels were significantly increased (* p =0.0243) in ∆gliT compared to wild-type. Tryprostatin B (E) and tryprostatin A (F) levels were significantly reduced in ∆gliT compared to wild-type (* p =0.0453 and ** p =0.008, respectively).
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Fig7: Levels of fumagillin, pseurotin A, fumitremorgin C, tryprostatin B and tryprostatin A inA. fumigatuswild-type and ∆gliTfollowing 96 h growth under secondary metabolite inducing conditions. Fumagillin (A) and pseurotin A (B) production was significantly reduced in A. fumigatus ∆gliT compared to wild-type (*p =0.0471 and * p =0.0297, respectively). There was no significant difference in fumitremorgin C (C) levels, while brevianamide F (D) levels were significantly increased (* p =0.0243) in ∆gliT compared to wild-type. Tryprostatin B (E) and tryprostatin A (F) levels were significantly reduced in ∆gliT compared to wild-type (* p =0.0453 and ** p =0.008, respectively).

Mentions: Of the 69 genes in the “supercluster” on chromosome 8, expression of two genes is significantly down-regulated in A. fumigatus wild-type in response to exogenous gliotoxin (Table 2). However, in A. fumigatus ∆gliT, when exposed to exogenous gliotoxin, expression of 26 genes from the “supercluster” was down-regulated (Table 2). Closer inspection of the fumitremorgin B biosynthetic genes revealed that expression of the non-ribosomal peptide synthetase (NRPS), A. fumigatus ftmA[29], was significantly down-regulated (log2 2.78-fold) in A. fumigatus wild-type in exogenous gliotoxin presence (Table 2). While in A. fumigatus ∆gliT, ftmA (log2 6.65-fold), ftmC (log2 5.68-fold), ftmD (log2 5.87-fold), ftmG (log2 6.52-fold) and ftmI (log2 1.45-fold) expression was significantly down-regulated, and ftmE expression was completely inhibited in response to exogenous gliotoxin (Table 2). qRT-PCR analysis of A. fumigatus ftmA confirmed decreased expression in both wild-type and ∆gliT following exposure to exogenous gliotoxin (Figure 5). Determination of the levels of the fumitremorgins and associated compounds in A. fumigatus wild-type and ∆gliT cultured in secondary metabolite-inducing conditions (96 h in Czpaeks-Dox Broth) revealed significant alterations in the levels of a number of cognate metabolites. Specifically, brevianamide F levels were significantly increased in A. fumigatus ∆gliT compared to wild-type (p =0.0243), while the levels of both tryprostatin A and tryprostatin B were significantly decreased (p =0.008 and p =0.0453) in A. fumigatus ∆gliT compared to wild-type (Figure 7). There was no significant difference determined in the level of fumitremorgin C between A. fumigatus wild-type and ∆gliT (Figure 7).Figure 7


RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

O'Keeffe G, Hammel S, Owens RA, Keane TM, Fitzpatrick DA, Jones GW, Doyle S - BMC Genomics (2014)

Levels of fumagillin, pseurotin A, fumitremorgin C, tryprostatin B and tryprostatin A inA. fumigatuswild-type and ∆gliTfollowing 96 h growth under secondary metabolite inducing conditions. Fumagillin (A) and pseurotin A (B) production was significantly reduced in A. fumigatus ∆gliT compared to wild-type (*p =0.0471 and * p =0.0297, respectively). There was no significant difference in fumitremorgin C (C) levels, while brevianamide F (D) levels were significantly increased (* p =0.0243) in ∆gliT compared to wild-type. Tryprostatin B (E) and tryprostatin A (F) levels were significantly reduced in ∆gliT compared to wild-type (* p =0.0453 and ** p =0.008, respectively).
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Fig7: Levels of fumagillin, pseurotin A, fumitremorgin C, tryprostatin B and tryprostatin A inA. fumigatuswild-type and ∆gliTfollowing 96 h growth under secondary metabolite inducing conditions. Fumagillin (A) and pseurotin A (B) production was significantly reduced in A. fumigatus ∆gliT compared to wild-type (*p =0.0471 and * p =0.0297, respectively). There was no significant difference in fumitremorgin C (C) levels, while brevianamide F (D) levels were significantly increased (* p =0.0243) in ∆gliT compared to wild-type. Tryprostatin B (E) and tryprostatin A (F) levels were significantly reduced in ∆gliT compared to wild-type (* p =0.0453 and ** p =0.008, respectively).
Mentions: Of the 69 genes in the “supercluster” on chromosome 8, expression of two genes is significantly down-regulated in A. fumigatus wild-type in response to exogenous gliotoxin (Table 2). However, in A. fumigatus ∆gliT, when exposed to exogenous gliotoxin, expression of 26 genes from the “supercluster” was down-regulated (Table 2). Closer inspection of the fumitremorgin B biosynthetic genes revealed that expression of the non-ribosomal peptide synthetase (NRPS), A. fumigatus ftmA[29], was significantly down-regulated (log2 2.78-fold) in A. fumigatus wild-type in exogenous gliotoxin presence (Table 2). While in A. fumigatus ∆gliT, ftmA (log2 6.65-fold), ftmC (log2 5.68-fold), ftmD (log2 5.87-fold), ftmG (log2 6.52-fold) and ftmI (log2 1.45-fold) expression was significantly down-regulated, and ftmE expression was completely inhibited in response to exogenous gliotoxin (Table 2). qRT-PCR analysis of A. fumigatus ftmA confirmed decreased expression in both wild-type and ∆gliT following exposure to exogenous gliotoxin (Figure 5). Determination of the levels of the fumitremorgins and associated compounds in A. fumigatus wild-type and ∆gliT cultured in secondary metabolite-inducing conditions (96 h in Czpaeks-Dox Broth) revealed significant alterations in the levels of a number of cognate metabolites. Specifically, brevianamide F levels were significantly increased in A. fumigatus ∆gliT compared to wild-type (p =0.0243), while the levels of both tryprostatin A and tryprostatin B were significantly decreased (p =0.008 and p =0.0453) in A. fumigatus ∆gliT compared to wild-type (Figure 7). There was no significant difference determined in the level of fumitremorgin C between A. fumigatus wild-type and ∆gliT (Figure 7).Figure 7

Bottom Line: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated.Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin.Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, National University of Ireland Maynooth, Maynooth, Co, Kildare, Ireland. sean.doyle@nuim.ie.

ABSTRACT

Background: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin.

Results: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin.

Conclusions: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.

Show MeSH
Related in: MedlinePlus