Nitrogen Catabolite Repression in members of Paracoccidioides complex

Highlights

• In Paracoccidioides, AreA GATA transcriptional factor is essential for alleviation of Nitrogen Catabolite Repression (NCR).

• Genes putatively involved in NCR response are demonstrated in Paracoccidioides and overview of NCR pathway are constructed.

• Alleviation of NCR conditions was established in Paracoccidioides in proline supplementation.

• The NCR mechanism can be important to biology and/or fungal pathogenesis.

Abstract

Paracoccidioides complex is a genus that comprises pathogenic fungi which are responsible by systemic disease Paracoccidioidomycosis. In host tissues, pathogenic fungi need to acquire nutrients in order to survive, making the uptake of nitrogen essential for their establishment and dissemination. Nitrogen utilization is employed by the alleviation of Nitrogen Catabolite Repression (NCR) which ensures the use of non-preferential or alternative nitrogen sources when preferential sources are not available. NCR is controlled by GATA transcription factors which act through GATA binding sites on promoter regions in NCR-sensitive genes. This process is responsible for encoding proteins involved with the scavenge, uptake and catabolism of a wide variety of non-preferential nitrogen sources. In this work, we predict the existence of AreA GATA transcription factor and feature the zinc finger domain by three-dimensional structure in Paracoccidioides. Furthermore, we demonstrate the putative genes involved with NCR response by means of in silico analysis. The gene expression profile under NCR conditions was evaluated. Demonstrating that P. lutzii supported transcriptional regulation and alleviated NCR in non-preferential nitrogen-dependent medium. The elucidation of NCR in members of Paracoccidioides complex will provide new knowledge about survival, dissemination and virulence for these pathogens with regard to nitrogen-scavenging strategies in the interactions of host-pathogens.

Introduction

Activation of Nitrogen Catabolite Repression (NCR) allows microorganisms the use of non-preferential nitrogen sources such as peptides, nitrates, nitrites, purines, protein, free amino acids and other nitrogen compounds when preferential nitrogen sources such as glutamine and/or ammonium are not available [1]. Inside the cell, the NCR mechanism is regulated by the expression of genes which encode proteins involved in the scavenge, uptake and catabolism of a wide variety of poor, alternative or non-preferential nitrogen sources [2]. Previously, this mechanism has been investigated in model organisms such as Aspergillus nidulans [3], Neurospora crassa [4,5] and Saccharomyces cerevisiae [6]. NCR is regulated by intrinsic mechanisms inside cells that are dependent on both outside and inside nitrogen levels. In this sense, the regulatory NCR control inside the cell has led to a growing interest in knowledge about pathogenic fungi due to their parasitism and their ability to utilize a wide range of nitrogen sources inside the host tissues [7].

The main transcription factors involved in NCR-sensitive gene expression belong to the GATA family activators [8]. GATA transcription factors bind to the DNA through zinc finger motifs to control transcription events in response to nitrogen environmental stimuli. In filamentous fungus and yeasts, these GATA transcription factors are responsible for NCR control, allowing the utilization of alternative or non-preferential nitrogen sources [9,10]. GATA transcription factors have a highly conserved DNA-binding domain consisting of a Cys2-Cys2 type zinc finger which possesses a high affinity to the consensus HGATAR sequences at the promoter region of NCR-sensitive genes [[10], [11], [12]].

In model organisms such as S. cerevisiae, GATA transcription factors Gln3 and Gat1 (Nil1) are responsible for the activation of NCR. They are also responsible for the expression of a large number of genes involved in nitrogen uptake and catabolism, such as general amino acid permease (GAP1), glutamine synthetase (GLN1), proline oxidase (PUT1), allantoate permease (DAL5) and NAD-specific glutamate dehydrogenase (GDH1) [[13], [14], [15]]. On the other hand, the GATA transcription factors Dal80p (Uga4p) and Deh1p (Nil2 or Gzf3) act in the repression of NCR-sensitive genes [2,14,16]. The action of GATA transcription factors Gln3 and Gat1 in the alleviation of NCR occurs through their binding at cis-acting nitrogen-regulated upstream activation sequence (UAS_NTR) elements in the promoter region of NCR-sensitive genes [17,18]. In addition, NCR in S. cerevisiae is also achieved through the nitrogen-response control of the TOR 1 pathway by rapamycin treatment [19,20].

In N. crassa and A. nidulans model organisms, the regulation of NCR genes is mediated by only positively-acting GATA transcription factors, Nit-2 and AreA respectively [1,21]. The NIT-2 and AREA genes are expressed in basal levels; however in the presence of non-preferential nitrogen sources gene expression increases and NCR is alleviated [22]. The A. nidulans AreA activity is tightly regulated at posttranscriptional and posttranslational levels where the mRNA stability of AreA transcript and AreA protein activity are involved with nitrogen levels [23]. The regulation and expression of the NIT-2 gene differs from the AREA gene. Thus, at the posttranscriptional level, Nit-2 mRNA is found in high levels even under nitrogen-rich conditions, and increased expression levels occur when conditions change for nitrogen starvation [22].

Repression of NCR control in A. nidulans and N. crassa acts as a trigger for the interaction between the GATA transcription factor and NMR protein (Nitrogen Metabolic Regulation). In N. crassa, Nit-2 function is impaired by protein-protein interaction with co-repressor Nmr1 which binds to the carboxy terminus tail of the Nit-2 protein [24]. Similar mechanisms of negative regulation are encountered in A. nidulans AreA, which are modulated by NmrA protein, an N. crassa NMR ortholog protein [[25], [26], [27]]. The negative AreB GATA transcription factor in A. nidulans presents a N-terminal GATA zinc finger and a C-terminal leucine zipper domains. These domains are responsible for modulating AreA's function probably by competing for the same binding sites into NCR-genes [8,28].

Non-pathogenic fungi also possess GATA factors, for example AreA in Aspergillus oryzae [29] and Aspergillus parasiticus [30], Nmc in Penicillium roqueforti [31] and Gaf1 in Schizosaccharomyces pombe [32]. Interestingly, in pathogenic fungus, GATA factors such as Nit2 in Ustilago maydis [33], Gln3p and Gat1p in Candida albicans [[34], [35]] and Gat1 in Cryptococcus neoformans [36] have been required for virulence traits.

Paracoccidioides genus is composed of five species: P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis and P. lutzii. All these species are causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America, with higher incidence in Brazil, Colombia, Venezuela and Ecuador [37,38]. The saprophytic form of the Paracoccidioides species is found in soil as mycelium. Under the influence of different conditions, such as temperature, humidity and/or nutrient availability, it develops into the germinative/infective spores known as conidia [39,40]. Inhalation of these propagules by animals or humans triggers the dimorphic transition to the pathogenic yeast form, resulting in the development of PCM disease. The highest incidences of PCM mycoses are encountered in the lungs, however this fungus can spread to other organs and tissues, developing multifocal forms of disease manifestation [[41], [42], [43]]. PCM mycoses represent an important public health risk, evidenced by the fact that the disease is the eighth highest cause of mortality among fungal infections and chronic diseases [[44], [45], [46], [47]].

Paracoccidioides are pathogenic fungi encountered in human hosts and wild animals [48], and can survive inside these hosts for several years [49]. Micro-nutritional acquisition is important for fungus survival and our group has been extensively investigating this aspect, with a focus on metal homeostasis and its role in pathogenicity and virulence [[50], [51], [52], [53]]. The macro-nutritional status in Paracoccidioides spp. has been explored by Lima et al. [54], with a focus on carbon starvation. However, the macro-nutritional status caused by nitrogen starvation has been poorly investigated in Paracoccidioides genus.

Taking the above into account, the aim of this study was to characterize GATA transcription factors involved with NCR alleviation in members of Paracoccidioides complex and highlight genes that can be involved in NCR control by the cell. NCR has been related to nitrogen uptake, virulence and survival in nitrogen-poor or nitrogen-starved environments. In turn, the elucidation of NCR in our model will open novel perspectives about nitrogen uptake during interactions with host-pathogens, as well as the understanding of the biology, adaptation and virulence strategies of Paracoccidioides.