Hydroxamate production as a high affinity iron acquisition mechanism in Paracoccidioides Spp.

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity

Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciensmediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms

The Homeostasis of Iron, Copper, and Zinc in Paracoccidioides Brasiliensis, Cryptococcus Neoformans Var. Grubii, and Cryptococcus Gattii: A Comparative Analysis

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. brasiliensis Pb01, 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

Zinc at the Host–Fungus Interface: How to Uptake the Metal?

Zinc is an essential nutrient for all living organisms. However, firm regulation must be maintained since micronutrients also can be toxic in high concentrations. This notion is reinforced when we look at mechanisms deployed by our immune system, such as the use of chelators or membrane transporters that capture zinc, when threatened with pathogens, like fungi. Pathogenic fungi, on the other hand, also make use of a variety of transporters and specialized zinc captors to survive these changes. In this review, we sought to explain the mechanisms, grounded in experimental analysis and described to date, utilized by pathogenic fungi to maintain optimal zinc levels

Paracoccidioides spp .ferrous and ferric iron assimilation pathways

Submitted by Jaqueline Silva ([email protected]) on 2018-05-14T19:24:11Z No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-05-15T13:18:32Z (GMT) No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-05-15T13:18:32Z (GMT). No. of bitstreams: 2 Artigo - Elisa Flávia Luiz Cardoso Bailão - 2015.pdf: 2352117 bytes, checksum: ad80fe6f836e11b501d4c14b1c2ac915 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-08Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation

Zinc Starvation Induces Cell Wall Remodeling and Activates the Antioxidant Defense System in <i>Fonsecaea pedrosoi</i>

The survival of pathogenic fungi in the host after invasion depends on their ability to obtain nutrients, which include the transition metal zinc. This essential micronutrient is required to maintain the structure and function of various proteins and, therefore, plays a critical role in various biological processes. The host’s nutritional immunity limits the availability of zinc to pathogenic fungi mainly by the action of calprotectin, a component of neutrophil extracellular traps. Here we investigated the adaptive responses of Fonsecaea pedrosoi to zinc-limiting conditions. This black fungus is the main etiological agent of chromoblastomycosis, a chronic neglected tropical disease that affects subcutaneous tissues. Following exposure to a zinc-limited environment, F. pedrosoi induces a high-affinity zinc uptake machinery, composed of zinc transporters and the zincophore Pra1. A proteomic approach was used to define proteins regulated by zinc deprivation. Cell wall remodeling, changes in neutral lipids homeostasis, and activation of the antioxidant system were the main strategies for survival in the hostile environment. Furthermore, the downregulation of enzymes required for sulfate assimilation was evident. Together, the adaptive responses allow fungal growth and development and reveals molecules that may be related to fungal persistence in the host

Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the Paracoccidioides genus

Submitted by Jaqueline Silva ([email protected]) on 2018-05-08T12:14:22Z No. of bitstreams: 2 Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-05-09T11:51:54Z (GMT) No. of bitstreams: 2 Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Rejected by Luciana Ferreira ([email protected]), reason: Checar os nomes dos autores no lattes , alterar o país e rever os espaços na citação. on 2018-05-09T12:11:26Z (GMT)Submitted by Jaqueline Silva ([email protected]) on 2018-05-14T18:37:34Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5)Rejected by Luciana Ferreira ([email protected]), reason: O país não é Brasil. A citação apresenta problemas de espaço: TRISTÂO, Gabriel B.et al. Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the Paracoccidioides genus. Frontiers in Microbiology, Lausanne, v.(ESPAÇO)5, n.(ESPAÇO)761, p.(ESPAÇO)1-11, jan. 2015. on 2018-05-15T13:11:50Z (GMT)Submitted by Jaqueline Silva ([email protected]) on 2018-05-15T19:30:42Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5)Rejected by Luciana Ferreira ([email protected]), reason: Olhe a NBR 6023 como se abrevia os meses em outros idiomas e a questão de espaço depois de v. e n. (semre deice um espaço para depois digitar o número: ERRADO Tv.5, n.761, p.1-11, jan. 2015. CERTO v. 5, n. 761, p.1-11, Jan. 2015. on 2018-05-16T11:42:00Z (GMT)Submitted by Jaqueline Silva ([email protected]) on 2018-05-23T19:24:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-05-28T12:07:21Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5)Made available in DSpace on 2018-05-28T12:07:21Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Artigo - Gabriel Brum Tristão - 2015.pdf: 2472227 bytes, checksum: 5bdf07b703f03b64021e19f11b883f4f (MD5) Previous issue date: 2015-01Approximately one-third of all proteins have been estimated to contain at least one metal cofactor, and these proteins are referred to as metalloproteins. These represent one of the most diverse classes of proteins, containing metal ions that bind to specific sites to perform catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Its function and the type of metal binder can also be predicted via a bioinformatics approach. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobic mycelia in the environment and as yeast, the parasitic form, in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. Many metalloproteins are important for the virulence of several pathogenic microorganisms. Accordingly, the present work aimed to predict the copper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides (Pb01, Pb03, and Pb18). The metalloproteins were identified using bioinformatics approaches based on structure, annotation and domains. Cu-, Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by Paracoccidioides spp. genomes. Zinc proteins were the most abundant metalloproteins, representing 5.7% of the fungus proteome, whereas copper and iron proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore, it was observed that many of these metalloproteins serve as virulence factors in the biology of the fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the Paracoccidioides spp. are of the utmost importance for the biology and virulence of these particular human pathogens

<i>Paracoccidioides</i> strains used in this study.

<p><i>Paracoccidioides</i> strains used in this study.</p

Iron limiting conditions induce biosynthesis and secretion of siderophores.

<p>A: Detection of siderophores in MMcM agar plates. Under iron sufficiency (30 µM Fe), the production of these chelators was repressed in solid medium. B: Percent siderophore activity was determined in <i>Pb</i>01and <i>Pb</i>18 supernatants. Values are expressed as mean <u>+</u> standard deviation of three experiments. *statistically significant difference determined by Student's <i>t</i>-test (p<0.05).</p