The influence of a copper efflux pump in Histoplasma capsulatum virulence.

During the infectious process, pathogenic microorganisms must obtain nutrients from the host in order to survive and proliferate. These nutritional sources include the metallic nutrient copper. Despite its essentiality, copper in large amounts is toxic. Host defense mechanisms use high copper poisoning as a fungicidal strategy to control infection. Transcriptional analyses showed that yeast cultured in the presence of copper or inside macrophages (24 h) had elevated expression of CRP1, a copper efflux pump, suggesting that Histoplasma capsulatum could be exposed to a high copper environment in macrophages during the innate immune stage of infection. Accordingly, macrophages cultured in high copper are more efficient in controlling H. capsulatum growth. Also, silencing of ATP7a, a copper pump that promotes the copper influx in phagosomes, increases fungal survival in macrophages. The rich copper environment faced by the fungus is not dependent on IFN-γ, since fungal CRP1 expression is induced in untreated macrophages. Appropriately, CRP1 knockdown fungal strains are more susceptible to macrophage control than wild-type yeasts. Additionally, CRP1 silencing decreases fungal burden in mice during the phase of innate immune response (4-day postinfection) and CRP1 is required for full virulence in a macrophage cell lines (J774 A.1 and RAW 264.7), as well as primary cells (BMDM). Thus, induction of fungal copper detoxifying genes during innate immunity and the attenuated virulence of CRP1-knockdown yeasts suggest that H. capsulatum is exposed to a copper-rich environment at early infection, but circumvents this condition to establish infection.

High copper promotes cell wall remodeling and oxidative stress in Histoplasma capsulatum, as revealed by proteomics.

Histoplasma experiences nutritional stress during infection as a result of immune cells manipulating essential nutrients, such as metal ions, carbon, nitrogen, and vitamins. Copper (Cu) is an essential metallic micronutrient for living organisms; however, it is toxic in excess. Microbial pathogens must resist copper toxicity to survive. In the case of Histoplasma, virulence is supported by high-affinity copper uptake during late infection, and copper detoxification machinery during early macrophage infection. The objective of this study was to characterize the global molecular adaptation of Histoplasma capsulatum to copper excess using proteomics. Proteomic data revealed that carbohydrate breakdown was repressed, while the lipid degradation pathways were induced. Surprisingly, the production of fatty acids/lipids was also observed, which is likely a result of Cu-mediated damage to lipids. Additionally, the data showed that the fungus increased the exposition of glycan and chitin on the cell surface in high copper. Yeast upregulated antioxidant enzymes to counteract ROS accumulation. The induction of amino acid degradation, fatty acid oxidation, citric acid cycle, and oxidative phosphorylation suggest an increase in aerobic respiration for energy generation. Thus, H. capsulatum's adaptive response to high Cu is putatively composed of metabolic changes to support lipid and cell wall remodeling and fight oxidative stress.

Fonsecaea pedrosoi produces ferricrocin and can utilize different host iron sources.

The survival of living organisms depends on iron, one of the most abundant metals in the Earth's crust. Nevertheless, this micronutrient is poorly available in our aerobic atmosphere as well as inside the mammalian host. This problem is circumvented by the expression of high affinity iron uptake machineries, including the production of siderophores, in pathogenic fungi. Here we demonstrated that F. pedrosoi, the causative agent of the neglected tropical disease chromoblastomycosis, presents gene clusters for siderophore production. In addition, ten putative siderophore transporters were identified. Those genes are upregulated under iron starvation, a condition that induces the secretion of hydroxamates, as revealed by chrome azurol S assays. RP-HPLC and mass spectrometry analysis allowed the identification of ferricrocin as an intra- and extracellular siderophore. F. pedrosoi can grow in different iron sources, including the bacterial ferrioxamine B and the host proteins ferritin, hemoglobin and holotransferrin. Of note, addition of hemoglobin, lactoferrin and holotransferrin to the growth medium of macrophages infected with F. pedrosoi enhanced significantly fungal survival. The ability to produce siderophores in iron limited conditions added to the versatility to utilize different sources of iron are strategies that certainly may contribute to fungal survival inside the host.

Iron Starvation Induces Ferricrocin Production and the Reductive Iron Acquisition System in the Chromoblastomycosis Agent Cladophialophora carrionii.

Iron is a micronutrient required by almost all living organisms. Despite being essential, the availability of this metal is low in aerobic environments. Additionally, mammalian hosts evolved strategies to restrict iron from invading microorganisms. In this scenario, the survival of pathogenic fungi depends on high-affinity iron uptake mechanisms. Here, we show that the production of siderophores and the reductive iron acquisition system (RIA) are employed by Cladophialophora carrionii under iron restriction. This black fungus is one of the causative agents of chromoblastomycosis, a neglected subcutaneous tropical disease. Siderophore biosynthesis genes are arranged in clusters and, interestingly, two RIA systems are present in the genome. Orthologs of putative siderophore transporters were identified as well. Iron starvation regulates the expression of genes related to both siderophore production and RIA systems, as well as of two transcription factors that regulate iron homeostasis in fungi. A chrome azurol S assay demonstrated the secretion of hydroxamate-type siderophores, which were further identified via RP-HPLC and mass spectrometry as ferricrocin. An analysis of cell extracts also revealed ferricrocin as an intracellular siderophore. The presence of active high-affinity iron acquisition systems may surely contribute to fungal survival during infection.

Global Molecular Response of Paracoccidioides brasiliensis to Zinc Deprivation: Analyses at Transcript, Protein and MicroRNA Levels.

Zinc is one of the main micronutrients for all organisms. One of the defense mechanisms used by the host includes the sequestration of metals used in fungal metabolism, such as iron and zinc. There are several mechanisms that maintain the balance in the intracellular zinc supply. MicroRNAs are effector molecules of responses between the pathogen and host, favoring or preventing infection in many microorganisms. Fungi of the Paracoccidioides genus are thermodimorphic and the etiological agents of paracoccidioidomycosis (PCM). In the current pandemic scenario world mycosis studies continue to be highly important since a significant number of patients with COVID-19 developed systemic mycoses, co-infections that complicated their clinical condition. The objective was to identify transcriptomic and proteomic adaptations in Paracoccidioides brasiliensis during zinc deprivation. Nineteen microRNAs were identified, three of which were differentially regulated. Target genes regulated by those microRNAs are elements of zinc homeostasis such as ZRT1, ZRT3 and COT1 transporters. Transcription factors that have zinc in their structure are also targets of those miRNAs. Transcriptional and proteomic data suggest that P. brasiliensis undergoes metabolic remodeling to survive zinc deprivation and that miRNAs may be part of the regulatory process.

Comparative proteomics in the three major human pathogenic species of the genus Sporothrix.

Sporotrichosis is a subcutaneous mycosis of humans and other mammals, caused by dimorphic species of the genus Sporothrix. In Brazil, human disease is broadly linked to transmission by infected cats and is mainly caused by Sporothrix brasiliensis, Sporothrix schenckii and Sporothrix globosa. In this study, we used a nanoscale liquid chromatography coupled with tandem mass spectrometry approach to provide the yeast proteomic profiles of S. brasiliensis, S. schenckii and S. globosa. From a total of 247 identified proteins, 137 were found as differentially expressed. Functional classification revealed that most are related to carbohydrate and amino acid metabolism as well as stress response. Our data indicate that S. brasiliensis metabolism is distinct of that of S. schenckii and S. globosa, mainly regarding amino acid metabolism and cell wall remodeling, which are induced in the former. Enzymes belonging to glycolytic pathway are, on the other hand, up-regulated in S. schenckii and S. globosa. These findings may explain the previously described more virulent character of S. brasiliensis. Besides complementing genomic comparisons already published, this first comparative proteomic study provided information that indicates new aspects of Sporothrix species metabolism as well as offers information that may be useful in the development of prospective functional studies.

Insights Into Histoplasma capsulatum Behavior on Zinc Deprivation.

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

Molecular characterization of siderophore biosynthesis in Paracoccidioides brasiliensis.

Iron is an essential nutrient for all organisms. For pathogenic fungi, iron is essential for the success of infection. Thus, these organisms have developed high affinity iron uptake mechanisms to deal with metal deprivation imposed by the host. Siderophore production is one of the mechanisms that fungal pathogens employ for iron acquisition. Paracoccidioides spp. present orthologous genes encoding the enzymes necessary for the biosynthesis of hydroxamates, and plasma membrane proteins related to the transport of these molecules. All these genes are induced in iron deprivation. In addition, it has been observed that Paracoccidioides spp. are able to use siderophores to scavenge iron. Here we observed that addition of the xenosiderophore ferrioxamine B FOB) to P. brasiliensis culture medium results in repression (at RNA and protein levels) of the SidA, the first enzyme of the siderophore biosynthesis pathway. Furthermore, SidA activity was reduced in the presence of FOB, suggesting that P. brasiliensis blocks siderophores biosynthesis and can explore siderophores in the environment to scavenge iron. In order to support the importance of siderophores on Paracoccidioides sp. life and infection cycle, silenced mutants for the sidA gene were obtained by antisense RNA technology. The obtained AsSidA strains displayed decreased siderophore biosynthesis in iron deprivation conditions and reduced virulence to an invertebrate model.

Propionate metabolism in a human pathogenic fungus: proteomic and biochemical analyses.

Fungi of the complex Paracoccidioides spp. are thermodimorphic organisms that cause Paracoccidioidomycosis, one of the most prevalent mycoses in Latin America. These fungi present metabolic mechanisms that contribute to the fungal survival in host tissues. Paracoccidioides lutzii activates glycolysis and fermentation while inactivates aerobic metabolism in iron deprivation, a condition found during infection. In lungs Paracoccidioides brasiliensis face a glucose poor environment and relies on the beta-oxidation to support energy requirement. During mycelium to yeast transition P. lutzii cells up-regulate transcripts related to lipid metabolism and cell wall remodeling in order to cope with the host body temperature. Paracoccidioides spp. cells also induce transcripts/enzymes of the methylcitrate cycle (MCC), a pathway responsible for propionyl-CoA metabolism. Propionyl-CoA is a toxic compound formed during the degradation of odd-chain fatty acids, branched chain amino acids and cholesterol. Therefore, fungi require a functional MCC for full virulence and the ability to metabolize propionyl-CoA is related to the virulence traits in Paracoccidioides spp. On this way we sought to characterize the propionate metabolism in Paracoccidioides spp. The data collected showed that P. lutzii grows in propionate and activates the MCC by accumulating transcripts and proteins of methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). Biochemical characterization of MCS showed that the enzyme is regulated by phosphorylation, an event not yet described. Proteomic analyses further indicate that P. lutzii yeast cells degrades lipids and amino acids to support the carbon requirement for propionate metabolism. The induction of a putative propionate kinase suggests that fungal cells use propionyl-phosphate as an intermediate in the production of toxic propionyl-CoA. Concluding, the metabolism of propionate in P. lutzii is under regulation at transcriptional and phosphorylation levels and that survival on this carbon source requires additional mechanisms other than activation of MCC.

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores.

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.

In vitro, ex vivo and in vivo models: A comparative analysis of Paracoccidioides spp. proteomic studies.

Members of the Paracoccidioides complex are human pathogens that infect different anatomic sites in the host. The ability of Paracoccidioides spp. to infect host niches is putatively supported by a wide range of virulence factors, as well as fitness attributes that may comprise the transition from mycelia/conidia to yeast cells, response to deprivation of micronutrients in the host, expression of adhesins on the cell surface, response to oxidative and nitrosative stresses, as well as the secretion of hydrolytic enzymes in the host tissue. Our understanding of how those molecules can contribute to the infection establishment has been increasing significantly, through the utilization of several models, including in vitro, ex vivo and in vivo infection in animal models. In this review we present an update of our understanding on the strategies used by the pathogen to establish infection. Our results were obtained through a comparative proteomic analysis of Paracoccidioides spp. in models of infection.

Identification of membrane proteome of Paracoccidioides lutzii and its regulation by zinc.

AIM: During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii. MATERIALS & METHODS: Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC-MS/MS detection and database search. RESULTS & CONCLUSION: Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides.

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 tumefaciens-mediated 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.

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

Approximately 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.