The homeostasis of iron, copper, and zinc in paracoccidioides brasiliensis, cryptococcus neoformans var. Grubii, and cryptococcus gattii: a comparative analysis

Abstract

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensisPb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

Keywords: infection; micronutrient homeostasis; pathogenic fungi.

Figure 1

Schematic comparison of iron metabolism in P. brasiliensis isolates and Cryptococcus species. Sit1p, MirAp, MirBp and MirCp are membrane transporters that traffic siderophores bound to ferric iron into the intracellular environment. SidAp, SidFp, SidCp and SidDp are enzymes from the biosynthetic pathway of hydroxamate-type siderophores. Ccc1p is a vacuolar membrane iron transporter. Mrs3/4p are iron transporters found in the inner mitochondrial membrane and Yfh1p is a mitochondrial matrix iron chaperone.

Figure 2

Domains found in a Frep related to iron reductive uptake in P. brasiliensis isolates and Cryptococcus species. The found domains are: ferric reductase domain (black boxes), FAD-binding domain (dark gray boxes) and NAD-binding domain (light gray boxes). The length of each protein, in amino acids, is shown on the right. Accession numbers: Sc (NP_014489), Pb01 (PAAG_06164.1), Pb03 (PABG_06497.1), Pb18 (PADG_07957.1), Cn (CNAG_00876.2) and Cg (CNBG_6082.2).

Figure 3

Conserved features in proteins involved in biosynthesis and uptake of siderophores. (A) Modular organization of SidCp in P. brasiliensis isolates. Black boxes represent adenylation (A) domain, dark gray boxes illustrate the condensation (C) domain and light gray boxes represent the peptidyl carrier (PCP) domain. The length of each protein, in amino acids, is shown on the right. (B) Topology of transmembrane domains of Sit1p from S. cerevisiae, P. brasiliensis isolates and Cryptococcus species. White boxes represent putative segments, according to cutoff parameters (cutoff for certain transmembrane segments 1.00; cutoff for putative transmembrane segments 0.60). E: extracellular environment; C: cytosol. The topology prediction was performed using the TopPred server. Accession numbers in A: Af (XP_753088), Pb01 (PAAG_08527.1), Pb03 (PABG_04670.1), Pb18 (PADG_05295.1). Accession numbers in B: Sc (NP_010849), Pb01 (PAAG_06516.1), Pb03 (PABG_02063.1), Pb18 (PADG_00462.1), Cn (CNAG_00815.2), and Cg (CNBG_1123.2).

Figure 4

Conserved features found in the primary structure of Ctr3p of P. brasiliensis isolates and Cryptococcus species. Ctr3p from P. brasiliensis isolates contains three putative transmembrane domains (TMD1-3, shown in black) while Ctr3p from Cryptococcus species presents only two TMDs. All species contain putative copper binding motifs (Mets motifs) arranged as MXXM and/or MXM. MXXXM motif in TMD3 in P. brasiliensis isolates and TMD2 in Cryptococcus species are represented in white characters. The length of each protein, in amino acids, is shown on the right. Accession numbers: Pb01 (PAAG_05251.1), Pb03 (PABG_07607.1), Pb18 (PADG_05084.1), Cn (CNAG_00979.2) and Cg (CNBG_0560.2).

Figure 5

Alignment of amino acid sequences of Zrt1p from S. cerevisiae, P. brasiliensis isolates and Cryptococcus species. The predicted transmembrane domains are shown in gray boxes. The black boxes inside the transmembrane segment contain conserved histidine-serine and glycine residues. The histidines found in the amino-terminal region of Zrt1p from Cryptococcus species and in the loop between transmembrane domains III and IV in P. brasiliensis and S. cerevisiae are boxed. Asterisks indicate amino acid identity and dots represent conserved substitutions. Accession numbers: Pb03 (PABG_07725.1), Pb18 (PADG_08567.1), Pb01 (PAAG_08727.1), Sc (NP_011259), Cn (CNAG_03398.2) and Cg (CNBG_2209.2).