The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans

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Highlights

  • LYS4 encoding homoaconitase in Cryptococcus neoformans was identified.

  • LYS4 expression is regulated by iron.

  • LYS4 plays critical roles in mitochondrial function, and antifungal sensitivity.

  • LYS4 is required for the full virulence in a mouse inhalation model of cryptococcosis.

Abstract

The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis.

Introduction

Lysine is an essential amino acid for animals and must be obtained from diet. Bacteria, fungi and plants, however, can synthesize lysine using two distinct pathways [1], [2]. In bacteria and plants, lysine is synthesized via the diaminopimelate (DAP) pathway, whereas the majority of fungi use the α-aminoadipate (AA) pathway [3]. The DAP pathway has been well studied in bacteria, and is considered a novel antibacterial target, especially in light of the absence of lysine biosynthesis in mammalian hosts [4]. Similarly, the AA pathway in fungi has been proposed as a valuable target for antifungal therapy. Indeed, several researchers have developed antifungal agents that specifically inhibit the AA pathway in pathogenic fungi [5], [6], [7].

In this study, we identified the gene encoding the putative homoaconitase in the human fungal pathogen Cryptococcus neoformans, which causes pneumonia and life-threatening meningoencephalitis, mainly in immunocompromised patients [8]. Homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthesis pathway [9]. The orthologous gene encoding homoaconitase, LYS4, in the model yeast Saccharomyces cerevisiae, is essential for lysine biosynthesis [10]. The protein encoded by S. cerevisiae LYS4 contains amino acid residues associated with an iron-sulfur (Fe–S) cluster and shows evolutionary conservation with the aconitase family of proteins [3], [11]. The S. cerevisiae ortholog of LYS4 in C. neoformans was initially found in our transcriptome analysis to identify differentially expressed genes in a mutant lacking CFO1, the gene encoding a ferroxidase for high-affinity iron transport at the plasma membrane [12]. The expression of LYS4 was significantly down-regulated in the cfo1 mutant, suggesting that Lys4 function may be associated with iron metabolism in C. neoformans. The fact that iron metabolism and homeostasis have a large influence on the virulence of C. neoformans [13], and that Lys4 is a possible antifungal drug target, led us to characterize its functions and roles in the physiology and virulence of the pathogen.

Section snippets

Strains, growth conditions and expression assays

The C. neoformans var. grubii strains (serotype A; MATα) used in this study are listed in Table S1. The strains were maintained in yeast extract-bacto peptone (YPD) medium with 2.0% glucose or yeast nitrogen base (YNB; Sigma, Saint Louis, MO, USA) with 2.0% glucose. To test the phenotype of the lysine auxotroph, 0.2 mg/mL of lysine (Sigma, Saint Louis, MO, USA) was added to YNB medium. The YNB low iron medium was prepared as described [14]. Construction of the lys4 mutant, the reconstituted

LYS4 is required for lysine biosynthesis in C. neoformans

Our previous transcriptome data showed that the gene CNAG_02565 was significantly down-regulated in the cfo1 mutant, which suggested a possible connection between the gene function and the iron homeostasis and metabolism in C. neoformans [12]. The protein sequence of CNAG_02565 is highly homologous to Lys4, an enzyme in the lysine biosynthesis pathway in S. cerevisiae, with 64% similarity and 52% identity [21]. We therefore designated CNAG_02565 as LYS4 and constructed a mutant strain lacking

Discussion

In this study, we identified and characterized LYS4 in C. neoformans, and demonstrated that the gene is expressed only when sufficient iron is available. The major iron-regulatory transcription factors HapX and Hap3 negatively regulated the expression of LYS4 in the low iron condition. We hypothesize that C. neoformans might down-regulate LYS4 expression to adapt to iron deficiency in the host, perhaps as part of a broader remodeling of metabolism. Similar remodeling and an impact on amino acid

Acknowledgements

This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, funded by the Ministry of Science, ICT, and Future Planning NRF-2013R1A1A1A05007037 (WJ), and by the National Institute of Allergy and Infectious Diseases RO1 AI053721 (JWK).

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