A Transcriptional Regulatory Map of Iron Homeostasis Reveals a New Control Circuit for Capsule Formation in Cryptococcus neoformans

Abstract

Iron is essential for the growth of the human fungal pathogen Cryptococcus neoformans within the vertebrate host, and iron sensing contributes to the elaboration of key virulence factors, including the formation of the polysaccharide capsule. C. neoformans employs sophisticated iron acquisition and utilization systems governed by the transcription factors Cir1 and HapX. However, the details of the transcriptional regulatory networks that are governed by these transcription factors and connections to virulence remain to be defined. Here, we used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome analysis (RNA-seq) to identify genes directly regulated by Cir1 and/or HapX in response to iron availability. Overall, 40 and 100 genes were directly regulated by Cir1, and 171 and 12 genes were directly regulated by HapX, under iron limited and replete conditions, respectively. More specifically, we found that Cir1 directly controls the expression of genes required for iron acquisition and metabolism, and indirectly governs capsule formation by regulating specific protein kinases, a regulatory connection not previously revealed. HapX regulates the genes responsible for iron-dependent pathways, particularly under iron-depleted conditions. By analyzing target genes directly bound by Cir1 and HapX, we predicted the binding motifs for the transcription factors and verified that the purified proteins bind these motifs in vitro. Furthermore, several direct target genes were coordinately and reciprocally regulated by Cir1 and HapX, suggesting that these transcription factors play conserved roles in the response to iron availability. In addition, biochemical analyses revealed that Cir1 and HapX are iron-containing proteins, implying that the regulatory networks of Cir1 and HapX may be influenced by the incorporation of iron into these proteins. Taken together, our identification of the genome-wide transcriptional networks provides a detailed understanding of the iron-related regulatory landscape, establishes a new connection between Cir1 and kinases that regulate capsule, and underpins genetic and biochemical analyses that reveal iron-sensing mechanisms for Cir1 and HapX in C. neoformans.

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

“…Under high-iron conditions, Cir1 is more abundant, and the protein acts as a repressor for genes, including those involved in iron uptake and metabolism. In contrast, HapX mainly acts as a repressor under low-iron conditions to inhibit the expression of genes involved in iron-consuming functions [ 36 ]. In the current study, we found that the protein levels of both Cir1 and HapX were significantly reduced in the sod2 mutant.…”

“…In mammalian cells, overexpression of mitochondrial Mn-SOD significantly reduced non-heme iron levels, which is in agreement with our data [ 37 , 38 ]. We hypothesize that increased superoxide due to lack of Sod2 may also cause a reduction in the stability and activity of Cir1 and HapX since the proteins are predicted to contain Fe-S clusters and iron influences their stability [ 21 , 36 ]. Indeed, previous studies have reported that superoxide causes the release of iron from the Fe-S cluster and in turn inactivates the Fe-S cluster-containing protein [ 39 , 40 ].…”

Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX

“…However, hapX and cir1 , a homologue of sreA , in Cryptococcus neoformans have been extensively studied in relation to virulence in humans [ 12 , 13 ]. Similar to the ascomycetes, hapX in C. neoformans represses iron utilization and promotes iron uptake and cir1 expression under iron starvation conditions [ 12 , 13 ]. Therefore, the deprivation of iron is a prerequisite for the expression of hapX and thus for the production of siderophore in fungi.…”

Yap5 Competes With Hap4 for the Regulation of Iron Homeostasis Genes in the Human Pathogen Candida glabrata

“…Upon iron deprivation, the HapX-CBC complex acts as a repressor of genes involved in irondependent pathways, i.e. genes encoding iron-sulfur cluster and heme containing proteins or involved in the biogenesis and assembly of these two prosthetic groups, and genes encoding transporters responsible for vacuolar sequestration of iron (Hortschansky et al, 2007;Jung et al, 2010;Singh et al, 2011;Lopez-Berges et al, 2012;Kröber et al, 2016;Wang et al, 2019;Do et al, 2020). In the human and plant pathogens Aspergillus fumigatus, Fusarium oxysporum and Verticillium Dahliae, HapX is also responsible for the induction of some of these "iron consuming genes" in high iron conditions, therefore playing a key role in iron tolerance (Gsaller et al, 2014;Wang et al, 2018).…”

“…HapX and its orthologues are transcriptionally induced by iron starvation and sense iron through conserved cysteine rich domains (CRD) in their C-terminal part (Hortschansky et al, 2007;Schrettl et al, 2010;Hsu et al, 2011;Singh et al, 2011;Lopez-Berges et al, 2012;Gsaller et al, 2014;Rietzschel et al, 2015;Kröber et al, 2016;Do et al, 2020). Importantly, HapX also contains a basic leucine zipper (bZIP) domain and belongs to the Yeast AP-1 (YAP) family of specific DNA binding transcriptional regulators (Tanaka et al, 2002;Singh et al, 2011;Rodrigues-Pousada et al, 2019).…”