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Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence.

ABSTRACT: Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complemented mutant. Both growth and germination rates of the mutant are reduced, and there are morphological abnormalities in conidiophores, leading to reduced conidiation. Conidia from the DeltapkaR mutant are more sensitive to killing by hydrogen peroxide, menadione, paraquat, and diamide. However, the hyphae of the mutant are killed to a greater extent only by paraquat and diamide, whereas they are less susceptible to the effects of hydrogen peroxide. In an immunosuppressed mouse model, intranasally administered conidia of the mutant are significantly less virulent than those of the wild type or a complemented mutant. Unregulated PKA signaling is detrimental to the virulence of A. fumigatus, perhaps through the reduced susceptibility of the mutant to damage by oxidizing agents and reduced growth kinetics.

SUBMITTER: Zhao W

PROVIDER: S-EPMC1539607 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence.

Zhao Wei W Panepinto John C JC Fortwendel Jarrod R JR Fox Lauren L Oliver Brian G BG Askew David S DS Rhodes Judith C JC

Infection and immunity 20060801 8

Aspergillus fumigatus is an important opportunistic fungal pathogen. The cAMP-dependent protein kinase (PKA) signaling pathway plays an important role in regulating morphology, growth, and virulence in a number of fungal pathogens of plants and animals. We have constructed a mutant of A. fumigatus that lacks the regulatory subunit of PKA, pkaR, and analyzed the growth and development, sensitivity to oxidative damage, and virulence of the mutant, along with those of the wild type and a complement ...[more]

PMID: 16861675

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Project description:The eukaryotic multisubunit Elongator complex has been shown to perform multiple functions in transcriptional elongation, histone acetylation and tRNA modification. However, the Elongator complex plays different roles in different organisms, and the underlying mechanisms remain unexplored. Moreover, the biological functions of the Elongator complex in human fungal pathogens remain unknown. In this study, we verified that the Elongator complex of the opportunistic fungal pathogen Aspergillus fumigatus consists of six subunits (Elp1-6), and the loss of any subunit results in similarly defective colony phenotypes with impaired hyphal growth and reduced conidiation. The catalytic subunit-Elp3 of the Elongator complex includes a S-adenosyl methionine binding (rSAM) domain and a lysine acetyltransferase (KAT) domain, and it plays key roles in the hyphal growth, biofilm-associated exopolysaccharide galactosaminogalactan (GAG) production, adhesion and virulence of A. fumigatus; however, Elp3 does not affect H3K14 acetylation levels in vivo. LC-MS/MS chromatograms revealed that loss of Elp3 abolished the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNA wobble uridine (U34), and the overexpression of tRNAGlnUUG and tRNAGluUUC, which normally harbor mcm5s2U modifications, mainly rescues the defects of the Δelp3 mutant, suggesting that tRNA modification rather than lysine acetyltransferase is responsible for the primary function of Elp3 in A. fumigatus. Strikingly, global proteomic comparison analyses showed significantly upregulated expression of genes related to amino acid metabolism in the Δelp3 mutant strain compared to the wild-type strain. Western blotting showed that deletion of elp3 resulted in overexpression of the amino acid starvation-responsive transcription factor CpcA, and deletion of CpcA markedly reversed the defective phenotypes of the Δelp3 mutant, including attenuated virulence. Therefore, the findings of this study demonstrate that A. fumigatus Elp3 functions as a tRNA-modifying enzyme in the regulation of growth, GAG production, adhesion and virulence by maintaining intracellular amino acid homeostasis. More broadly, our study highlights the importance of U34 tRNA modification in regulating cellular metabolic states and virulence traits of fungal pathogens.

Project description:The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. Previous work showed that in A. fumigatus, the Lim domain-binding protein PtaB can form a complex with the sequence-specific transcription factor SomA for regulating GAG biosynthesis, biofilm formation, and asexual development. However, transcriptional coactivators required for biofilm formation in A. fumigatus remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in the GAG biosynthesis, biofilm formation, conidiation, and virulence of A. fumigatus. RNA sequence data demonstrated that Spt20 positively regulates the expression of the GAG biosynthesis genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex (known from yeast) could be immunoprecipitated with Spt20, suggesting that Spt20 acts as a structural subunit of the SAGA complex. Furthermore, distinct modules of SAGA regulate GAG biosynthesis, biofilm formation, and asexual development in A. fumigatus to various degrees. In summary, the novel biofilm regulator Spt20 is reported, which plays a crucial role in the regulation of fungal asexual development, GAG biosynthesis, and virulence in A. fumigatus. These findings expand knowledge on the regulatory circuits of the SAGA complex relevant for the biofilm formation and asexual development of A. fumigatus. IMPORTANCE Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors to transcriptional coactivators (chromatin regulators and the general transcription machinery) and their regulators. Previous research indicated that the sequence-specific complex SomA/PtaB regulates the biofilm formation and asexual development of Aspergillus fumigatus. However, transcriptional coactivators working with sequence-specific transcription factors to regulate A. fumigatus biofilm formation remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a novel regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in galactosaminogalactan (GAG) production, conidiation, and virulence. Moreover, nearly all modules of the SAGA complex were required for the biofilm formation and asexual development of A. fumigatus. These results establish the SAGA complex as a transcriptional coactivator required for the biofilm formation and asexual development of A. fumigatus.

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Dataset Information

Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence.

Publications

Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence.

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