mSphere of Influence: the Importance of Metabolism for Pathogen Adaptation to Host-Imposed Stresses

ABSTRACT Jorge Amich studies several aspects of sulfur and nitrogen metabolism in Aspergillus fumigatus, with the ultimate aim of identifying targets for the development of novel antifungals. In this mSphere of Influence article, he reflects on how “Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis” (A. McDonagh, N. D. Fedorova, J. Crabtree, Y. Yu, S. Kim, et al., PLoS Pathog 4:e1000154, 2008, https://doi.org/10.1371/journal.ppat.1000154) impacted his thinking about in vivo metabolism and how to investigate it

Repression of the acid ZrfA/ZrfB zinc-uptake system of Aspergillus fumigatus mediated by PacC under neutral, zinc-limiting conditions

The ZrfA and ZrfB transporters are components of a zinc-uptake system of Aspergillus fumigatus that mainly operates under acidic, zinc-limiting conditions. Expression of the genes zrfA and zrfB is up-regulated by the transcriptional activator ZafA in both acidic and neutral, zinc-limiting media. The transcription of zafA is not influenced by PacC, which is the transcriptional regulator involved in regulating pH homeostasis in Aspergillus. However, at neutral pH the expression of both zrfA and zrfB is significantly reduced. In this work, the repression of zrfA and zrfB in neutral and alkaline, zinc-limiting media was found to be mediated by the transcriptional regulator PacC. [Int Microbiol 2009; 12(1):39-47

Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus

Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen

Mutant characterization and in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for Aspergillus fumigatus virulence

Pathogenicity of the saprobe Aspergillus fumigatus strictly depends on nutrient acquisition during infection, as fungal growth determines colonisation and invasion of a susceptible host. Primary metabolism has to be considered as a valid target for antimycotic therapy, based on the fact that several fungal anabolic pathways are not conserved in higher eukaryotes. To test whether fungal proliferation during invasive aspergillosis relies on endogenous biosynthesis of aromatic amino acids, defined auxotrophic mutants of A. fumigatus were generated and assessed for their infectious capacities in neutropenic mice and found to be strongly attenuated in virulence. Moreover, essentiality of the complete biosynthetic pathway could be demonstrated, corroborated by conditional gene expression in infected animals and inhibitor studies. This brief report not only validates the aromatic amino acid biosynthesis pathway of A. fumigatus to be a promising antifungal target but furthermore demonstrates feasibility of conditional gene expression in a murine infection model of aspergillosis

The Transcription Factor ZafA Regulates the Homeostatic and Adaptive Response to Zinc Starvation in Aspergillus fumigatus

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