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.
Histoplasma capsulatum elevates Crp1 expression in the presence of copper in vitro or in macrophage infection. Copper chelation hampered macrophage ability to control the fungus, while fungal burden decreases in copper‐treated immune cells. ATP7a silencing (phagosome copper pump) also caused increased fungal burden. H. capsulatum CRP1‐knockdown strains attenuated virulence. These data show that copper increase is a strategy for phagocytes to control H. capsulatum, and Crp1 is responsible for H. capsulatum virulence.
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.
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.
Iron 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, (59)Fe 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.
Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of ...treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.
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.
Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. ...In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.
The genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to ...fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis.
The fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MSE) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages.
For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche.
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.
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