Molecular aspects of copper homeostasis in fungi.

Copper homeostasis in fungi is a tightly regulated process crucial for cellular functions. Fungi acquire copper from their environment, with transporters facilitating its uptake into the cell. Once inside, copper is utilized in various metabolic pathways, including respiration and antioxidant defense. However, excessive copper can be toxic by promoting cell damage mainly due to oxidative stress and metal displacements. Fungi employ intricate regulatory mechanisms to maintain optimal copper levels. These involve transcription factors that control the expression of genes involved in copper transport, storage, and detoxification. Additionally, chaperone proteins assist in copper trafficking within the cell, ensuring its delivery to specific targets. Furthermore, efflux pumps help remove excess copper from the cell. Altogether, these mechanisms enable fungi to balance copper levels, ensuring proper cellular function while preventing toxicity. Understanding copper homeostasis in fungi is not only essential for fungal biology but also holds implications for various applications, including biotechnology and antifungal drug development.

GlyT1 Inhibition by NFPS Promotes Neuroprotection in Amyloid-ß-Induced Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-ß, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-ß-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24 h prior to intrahippocampal injection of amyloid-ß. NFPS pretreatment prevented amyloid-ß-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-ß injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6 h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-ß-induced hippocampal damage.

Zinc Starvation Induces Cell Wall Remodeling and Activates the Antioxidant Defense System in Fonsecaea pedrosoi.

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.

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.

Proteomic analysis reveals changes in the proteome of human THP-1 macrophages infected with Paracoccidioides brasiliensis.

Paracoccidioides spp. is the etiologic agent of Paracoccidioidomycosis (PCM), a systemic disease with wide distribution in Latin America. Macrophages are very important cells during the response to infection by P. brasiliensis. In this study, we performed a proteomic analysis to evaluate the consequences of P. brasiliensis yeast cells on the human THP-1 macrophage proteome. We have identified 443 and 2247 upregulated or downregulated proteins, respectively, in macrophages co-cultured with yeast cells of P. brasiliensis in comparison to control macrophages unexposed to the fungus. Proteomic analysis revealed that interaction with P. brasiliensis caused metabolic changes in macrophages that drastically affected energy production pathways. In addition, these macrophages presented regulated many factors related to epigenetic modifications and gene transcription as well as a decrease of many proteins associated to the immune system activity. This is the first human macrophage proteome derived from interactions with P. brasiliensis, which contributes to elucidating the changes that occur during the host response to this fungus. Furthermore, it highlights proteins that may be targets for the development of new therapeutic approaches to PCM.

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.

The State of the Art in Transcriptomics and Proteomics of Clinically Relevant Sporothrix Species.

Proteomics provide a robust approach to profile and quantify proteins within cells, organs, or tissues, providing comprehensive insights about the dynamics of cellular processes, modifications, and interactions. Similarly, understanding the transcriptome is essential to decipher functional elements of the genome, unraveling the mechanisms of disease development and the molecular constituents of cells and tissues. Some thermodimorphic fungi of the genus Sporothrix cause sporotrichosis, a subcutaneous mycosis of worldwide relevance. The transcriptome and proteome of the main Sporothrix species of clinical interest can elucidate the mechanisms underlying pathogenesis and host interactions. Studies of these techniques can contribute to the advancement of novel diagnostic and therapeutic strategies. A literature review was carried out, addressing all articles based on proteomics using mass spectrometry and transcriptomics of Sporothrix spp. Twenty-one studies were eligible for this review. The main findings include proteins and genes involved in dimorphism, cell differentiation, thermotolerance, virulence, immune evasion, metabolism, cell adhesion, cell transport, and biosynthesis. With the spread and emergence of sporotrichosis in different countries, ongoing research efforts and new discoveries are welcome to advance knowledge about this mycosis and its agents.

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.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

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.

New Methylcitrate Synthase Inhibitor Induces Proteolysis, Lipid Degradation and Pyruvate Excretion in Paracoccidioides brasiliensis.

BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by the inhalation of conidia of the genus Paracoccidioides. During the infectious process, fungal cells use several carbon sources, leading to the production of propionyl-CoA. The latter is metabolized by the methylcitrate synthase, a key enzyme of the methylcitrate cycle. We identified an inhibitor compound (ZINC08964784) that showed antifungal activity against P. brasiliensis. METHODS: This work aimed to understand the fungal metabolic response of P. brasiliensis cells exposed to ZINC08964784 through a proteomics approach. We used a glucose-free medium supplemented with propionate in order to simulate the environment found by the pathogen during the infection. We performed pyruvate dosage, proteolytic assay, dosage of intracellular lipids and quantification of reactive oxygen species in order to validate the proteomic results. RESULTS: The proteomic analysis indicated that the fungal cells undergo a metabolic shift due to the inhibition of the methylcitrate cycle and the generation of reactive species. Proteolytic enzymes were induced, driving amino acids into degradation for energy production. In addition, glycolysis and the citric acid cycle were down-regulated while ß-oxidation was up-regulated. The accumulation of pyruvate and propionyl-CoA led the cells to a state of oxidative stress in the presence of ZINC08964784. CONCLUSIONS: The inhibition of methylcitrate synthase caused by the compound promoted a metabolic shift in P. brasiliensis damaging energy production and generating oxidative stress. Hence, the compound is a promising alternative for developing new strategies of therapies against paracoccidioidomycosis.

Bioluminescence imaging in Paracoccidioides spp.: a tool to monitor the infectious processes.

The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM.

An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae).

Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

Comparative Proteomic Analysis of Histoplasma capsulatum Yeast and Mycelium Reveals Differential Metabolic Shifts and Cell Wall Remodeling Processes in the Different Morphotypes.

Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation was performed by enzymatic activity determination, Western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast cells. The proteomic profile-associated functional classification analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum.

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.

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.

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.

Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation.

Copper is an essential micronutrient for the performance of important biochemical processes such as respiration detoxification, and uptake of metals like iron. Studies have shown that copper deprivation is a strategy used by the host against pathogenic fungi such as Cryptoccocus neoformans and Candida albicans during growth and development of infections in the lungs and kidneys. Although there are some studies, little is known about the impact of copper deprivation in members of the Paracoccidioides genus. Therefore, using isobaric tag labeling (iTRAQ)-Based proteomic approach and LC-MS/MS, we analyzed the impact of in vitro copper deprivation in the metabolism of Paracoccidioides brasiliensis. One hundred and sixty-four (164) differentially abundant proteins were identified when yeast cells were deprived of copper, which affected cellular respiration and detoxification processes. Changes in cellular metabolism such as increased beta oxidation and cell wall remodeling were described.

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.