Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30.

BACKGROUND: Trichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported. RESULTS: Although high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1-4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase. CONCLUSIONS: For the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.

RNA-Seq Analysis of the Effect of Zinc Deficiency on Microsporum canis, ZafA Gene Is Important for Growth and Pathogenicity.

Microsporum canis, a common pathogenic skin fungus, can cause dermatophytosis in humans and animals. Zinc is an important trace element and plays an important role in the growth and metabolism of fungi. Currently, the effects of zinc deficiency on growth, gene expression, and metabolic pathway have not been clarified in M. canis. Therefore, M. canis was cultured under zinc restriction, and RNA-Seq was conducted in this study. The growth of M. canis was severely inhibited, and many genes showed significant upregulation and downregulation in M. canis with zinc deficiency. Zinc deficiency could negatively affect the gene expression and biological metabolic pathway in M. canis. The zinc-responsiveness transcriptional activator (ZafA) gene was significantly upregulated and shared homology with Zap1. Thus, the ZafA gene might be the main transcription factor regulating M. canis zinc homeostasis. The ZafA gene knockout strain, ZafA-hph, was constructed via Agrobacterium tumefaciens-mediated transformation (ATMT) in M. canis for the first time to assess its function. In vitro growth ability, hair biodegradation ability, virulence test, and zinc absorption capacity in ZafA-hph and wild-type M. canis strains were compared. Results showed that the ZafA gene plays an important role in zinc absorption, expression of zinc transporter genes, and growth and pathogenicity in M. canis and can be used as a new drug target. Cutting off the zinc absorption pathway can be used as a way to prevent and control infection in M. canis.

The Role of Zinc in Copper Homeostasis of Aspergillus fumigatus.

Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the Δafmac1 strain on AMM. In addition, the growth defect of the Δafmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the Δafmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the Δafmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5'-CAA(G)GGT-3' was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.

ZafA-mediated regulation of zinc homeostasis is required for virulence in the plant pathogen Fusarium oxysporum.

During infection, soilborne fungal pathogens face limiting conditions of different metal ions, including zinc. The role of zinc homeostasis in fungal pathogenicity on plants remains poorly understood. Here it is shown that the transcription factor ZafA, orthologous to Saccharomyces cerevisiae Zap1, functions as a key regulator of zinc homeostasis and virulence in Fusarium oxysporum, a cross-kingdom pathogen that causes vascular wilt on more than 100 plant species and opportunistic infections in humans. Expression of zafA is induced under zinc-limiting conditions and repressed by zinc. Interestingly, zafA is markedly up-regulated during early stages of plant infection, suggesting that F. oxysporum must cope with limited availability of zinc. Deletion of zafA results in deactivation of high-affinity zinc transporters, leading to impaired growth under zinc deficiency. Fusarium oxysporum strains lacking ZafA are reduced in their capability to invade and kill tomato plants and the non-vertebrate animal model Galleria mellonella. Collectively, the results indicate that ZafA-mediated adaptation to zinc deficiency is required for full virulence of F. oxysporum on plant and animal hosts.

The Role of Zinc in Gliotoxin Biosynthesis of Aspergillus fumigatus.

Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.

ZafA Gene Is Important for Trichophyton mentagrophytes Growth and Pathogenicity.

Trichophyton mentagrophytes is a common fungal pathogen that causes human and animal dermatophytosis. Previous studies have shown that zinc deficiency inhibits T. mentagrophytes growth, and the ZafA gene of T. mentagrophytes can code the functionally similar zinc finger transcriptional factor that can promote zinc ion absorption; however, the impact of ZafA on virulence and pathogenicity remains undetermined. To assess its gene function, the ZafA mutant, ZafA-hph, and the ZafA complemented strain, ZafA+bar, were constructed via Agrobacterium tumefaciens-mediated transformation. Polymerase chain reaction and Southern blot analyses were used to confirm the disruption. In vitro growth capacity and virulence analyses comparing ZafA-hph with wild-type T. mentagrophytes and ZafA+bar showed that ZafA-hph's growth performance, reproduction ability, and zinc ion absorption capacity were significantly lower than the wild-type T. mentagrophytes and ZafA+bar. ZafA-hph also showed weak hair biodegradation ability and animal pathogenicity. Thus, the significant decrease in T. mentagrophytes' growth ability and virulence was due to a lack of the zinc-responsive activity factor rather than the transformation process. This study confirmed that the T. mentagrophytes' zinc-responsive activity factor plays important roles in the pathogen's growth, reproduction, zinc ion absorption, and virulence. This factor is important and significant for effectively preventing and controlling T. mentagrophytes infections.