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A novel C2H2 transcription factor that regulates gliA expression interdependently with GliZ in Aspergillus fumigatus.

ABSTRACT: Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5' untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression.

SUBMITTER: Schoberle TJ

PROVIDER: S-EPMC4006717 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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A novel C2H2 transcription factor that regulates gliA expression interdependently with GliZ in Aspergillus fumigatus.

Schoberle Taylor J TJ Nguyen-Coleman C Kim CK Herold Jennifer J Yang Ally A Weirauch Matt M Hughes Timothy R TR McMurray John S JS May Gregory S GS

PLoS genetics 20140501 5

Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibi ...[more]

PMID: 24784729

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Project description:One of the most important features that enables Aspergillus fumigatus to grow within a susceptible individual and to cause disease is its ability to obtain Zn2+ ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in A. fumigatus relies on ZIP transporters encoded by the zrfA, zrfB and zrfC genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for A. fumigatus virulence. We combined the use of microarrays, Electrophoretic Mobility Shift Assays (EMSA) analyses, DNase I footprinting assays and in silico tools to better understand the regulation of the homeostatic and adaptive response of A. fumigatus to zinc starvation. We found that under zinc-limiting conditions, ZafA functions mainly as a transcriptional activator through binding to a zinc response sequence located in the regulatory regions of its target genes, although it could also function as a repressor of a limited number of genes. In addition to genes involved in the homeostatic response to zinc deficiency, ZafA also influenced, either directly or indirectly, the expression of many other genes. It is remarkable that the expression of many genes involved in iron uptake and ergosterol biosynthesis is strongly reduced under zinc starvation, even though only the expression of some of these genes appeared to be influenced directly or indirectly by ZafA. In addition, it appears to exist in A. fumigatus a zinc/iron cross-homeostatic network to allow the adaptation of the fungus to grow in media containing unbalanced Zn:Fe ratios. The adaptive response to oxidative stress typically linked to zinc starvation was also mediated by ZafA, as was the strong induction of genes involved in gliotoxin biosynthesis and self-protection against endogenous gliotoxin. This study has expanded our knowledge about the regulatory and metabolic changes displayed by A. fumigatus in response to zinc starvation and has helped us to pinpoint new ZafA target genes that could be important for fungal pathogens to survive and grow within host tissues and, hence, for virulence.

Project description:AimTo investigate the expression of macrophage migration inhibitory factor (MIF) and detect its role in the innate immune response of fungal keratitis (FK).MethodsWe collected the paraffin-embedded cornea tissues from 10 FK and 6 ocular trauma patients to explore the MIF expression by immunohistochemistry. Then we cultured telomease-immortalized human corneal epithelial cells (THCEs), stimulated by the hyphae suspension of Aspergillus fumigatus (A. fumigatus) to detect the change of MIF with or without the pretreatment of MIF inhibitor [4-Iodo-6-phenylpyrimidine (4-IPP)] by real-time polymerase chain reaction (PCR). The protein level of MIF was also tested by immunohistochemistry, and the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA were compared between normal, hyphae stimulated and 4-IPP pretreated groups by real-time PCR to study the influence of MIF on the expression of TNF-α and IL-6. Corneal severity of rats' FK models was documented by clinical scores, and real-time PCR. Western blot and immunohistochemistry were used to test the expression of MIF, TNF-α and IL-6 in rats' corneas.ResultsIn the corneas of FK patients, there was much stronger expression of MIF than that in the normal group showed by immunohistochemistry. In cultured THCEs stimulated by A. fumigatus, the expression of MIF became stronger in both immunohistochemistry and PCR at 16, 24, 32 and 48h post infection (p.i.; P<0.01, P<0.01, P<0.01, P<0.05). After pretreated with 4-IPP, the expression of MIF reduced at 4, 8, 16h p.i. (P<0.05, P<0.05, P<0.05) and the downstream TNF-α and IL-6 decreased obviously (P<0.05, P<0.01). In rats with A. fumigatus keratitis, the relative mRNA and protein level of MIF increased than those in the normal group by PCR (at 1d: P<0.01, 3d: P<0.01, 5d: P<0.01), Western blot and immunohistochemistry. After blocked MIF with 4-IPP, the clinical outcomes of rat keratitis showed markedly reduced inflammatory response (P<0.01), with TNF-α and IL-6 decreased in accordance with those in THCEs by PCR (P<0.05, P<0.01).ConclusionThe expression of MIF increased significantly in FK patients, THCEs and rats stimulated by A. fumigatus. After blocked with 4-IPP, the expression of MIF reduced, and so did its downstream cytokines: TNF-α and IL-6. The inflammation reaction of the rats' corneas lightened after pretreated with 4-IPP. MIF may play a role in the innate immune response of the corneal resistance against A. fumigatus.

Project description:The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA, brlA, and wetA were decreased in the ?rgdA mutant compared to those in the wild type (WT). Moreover, ?rgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the ?rgdA mutant. Comparative transcriptomic analyses revealed that the ?rgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the ?rgdA mutant exhibited elevated production of GT, ?rgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the ?rgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling.IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development.

Dataset Information

A novel C2H2 transcription factor that regulates gliA expression interdependently with GliZ in Aspergillus fumigatus.

Publications

A novel C2H2 transcription factor that regulates gliA expression interdependently with GliZ in Aspergillus fumigatus.

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