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The formamidase (FMD) enzyme plays an important role in fungal thriving by releasing a secondary nitrogen source as a product of its activity. In Paracoccidioides species, previous studies have demonstrated the upregulation of this enzyme in a wide range of starvation and infective-like conditions. However, Paracoccidioides lutzii formamidase has not yet been defined as a virulence factor. Here, by employing in vivo infections using an fmd-silenced strain in Galleria mellonella larvae model, we demonstrate the influence of formamidase in P. lutzii's immune stimulation and pathogenicity. The formamidase silencing resulted in improper arrangement of the nodules, poor melanogenesis and decreased fungal burden. Thus, we suggest that formamidase may be a piece composing the process of molecular recognition by Galleria immune cells. Furthermore, formamidase silencing doubled the observed survival rate of the larvae, demonstrating its importance in fungal virulence in vivo. Therefore, our findings indicate that formamidase contributes to Galleria's immune incitement and establishes the role of this enzyme as a P. lutzii virulence factor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pereira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MS E proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection.

This study describes the development of a simple, disposable, and eco-friendly electrochemical immunosensor for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Electrochemical devices were manufactured by stencil-printing using low-cost materials such as polyester sheets, graphite flakes, and natural resin. The immunosensor comprises gold nanoparticles stabilized with cysteamine, glutaraldehyde, anti-SARS-CoV-2 S protein monoclonal antibody (Ab1) as the biological receptor, and bovine serum albumin as a protective layer. The COVID-19 diagnostic was based on rapid square wave voltammetry measurements (15 min) using [Fe(CN) 6 ] 3-/4- as a redox probe. The method presented a linear response in the concentration range from 250 pg mL -1 to 20 μg mL -1 S protein, with a limit of detection of 36.3 pg mL -1 . The proposed immunosensor was stable for up to two weeks when stored at 4 °C and it demonstrated excellent clinical performance in diagnosing COVID-19 when applied to a panel of 44 undiluted swab samples collected from symptomatic patients. In comparison with results obtained through the quantitative reverse transcription polymerase chain reaction method, the proposed immunosensor offered 100 % accuracy, thus emerging as a powerful alternative candidate for routine and decentralized testing, which can be helpful in controlling the COVID-19 outbreak., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Urinary tract infections (UTIs) are common human infections that compromise women's health around the world, even though they can affect men and women of all ages. Bacterial species are the primary causative agents of UTIs, while Staphylococcus saprophyticus, a gram-positive bacterium, is especially important for uncomplicated infections in young women. Despite the number of antigenic proteins identified in Staphylococcus aureus and other bacteria of the genus, there is no immunoproteomic study in S. saprophyticus. In this context, since pathogenic microorganisms secrete important proteins that interact with hosts during infection, the present work aims to identify the exoantigens from S. saprophyticus ATCC 15305 by immunoproteomic and immunoinformatic approaches. We identified 32 antigens on the exoproteome of S. saprophyticus ATCC 15305 by immunoinformatic tools. By using 2D-IB immunoproteomic analysis, it was possible to identify 3 antigenic proteins: transglycosylase IsaA, enolase and the secretory antigen Q49ZL8. In addition, 5 antigenic proteins were detected by immunoprecipitation (IP) approach, where the most abundant were bifunctional autolysin and transglycosylase IsaA proteins. The transglycosylase IsaA was the only protein detected by all the tools approaches used in this study. In this work it was possible to describe a total of 36 S. saprophyticus exoantigens. Immunoinformatic analysis allowed the identification of 5 exclusive linear B cell epitopes from S. saprophyticus and 5 epitopes presenting homology with other bacteria that cause UTIs. This work describes, for the first time, the profile of exoantigens secreted by S. saprophyticus and can contribute to the identification of new diagnostic targets of UTIs, as well as to develop vaccines and immunotherapies against bacterial urinary infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Nitrogen is a crucial nutrient for microorganisms that compose essential biomolecules. However, hosts limit this nutrient as a strategy to counter infections, therefore, pathogens use adaptive mechanisms to uptake nitrogen from alternative sources. In fungi, nitrogen catabolite repression (NCR) activates transcription factors to acquire nitrogen from alternative sources when preferential sources are absent. Formamidase has been related to nitrogen depletion in Aspergillus nidulans through formamide degradation to use the released ammonia as a nitrogen source. In Paracoccidioides spp., formamidase is highly expressed in transcriptomic and proteomic analyses. Here, we aim to investigate the importance of formamidase to Paracoccidioides lutzii . Thereby, we developed a P. lutzii silenced strain of fmd gene (AsFmd) by antisense RNA technology using Agrobacterium tumefaciens -mediated transformation (ATMT). The AsFmd strain led to increased urease expression, an enzyme related to nitrogen assimilation in other fungi, suggesting that P. lutzii might explore urease as an alternative route for ammonia metabolism as a nitrogen source. Moreover, formamidase was important for fungal survival inside macrophages, as fungal recovery after macrophage infection was lower in AsFmd compared to wild-type (WT) strain. Our findings suggest potential alternatives of nitrogen acquisition regulation in P. lutzii , evidencing formamidase influence in fungal virulence.

Paracoccidioides complex is a genus that comprises pathogenic fungi which are responsible by systemic disease Paracoccidioidomycosis. In host tissues, pathogenic fungi need to acquire nutrients in order to survive, making the uptake of nitrogen essential for their establishment and dissemination. Nitrogen utilization is employed by the alleviation of Nitrogen Catabolite Repression (NCR) which ensures the use of non-preferential or alternative nitrogen sources when preferential sources are not available. NCR is controlled by GATA transcription factors which act through GATA binding sites on promoter regions in NCR-sensitive genes. This process is responsible for encoding proteins involved with the scavenge, uptake and catabolism of a wide variety of non-preferential nitrogen sources. In this work, we predict the existence of AreA GATA transcription factor and feature the zinc finger domain by three-dimensional structure in Paracoccidioides. Furthermore, we demonstrate the putative genes involved with NCR response by means of in silico analysis. The gene expression profile under NCR conditions was evaluated. Demonstrating that P. lutzii supported transcriptional regulation and alleviated NCR in non-preferential nitrogen-dependent medium. The elucidation of NCR in members of Paracoccidioides complex will provide new knowledge about survival, dissemination and virulence for these pathogens with regard to nitrogen-scavenging strategies in the interactions of host-pathogens., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paracoccidioidomycosis is a highly prevalent systemic mycosis in Latin America, caused by fungi of the genus Paracoccidioides. Copper is essential for eukaryotes and bacteria. This micronutrient is used in many vital biochemical processes, although metal excess levels can be toxic for organisms. Pathways underlying copper overload are poorly understood in members of the Paracoccidioides complex. The responses of Paracoccidioides lutzii yeast cells to copper overload were here evaluated. The results showed that under copper overload, cells presented a dark brown pigment, identified as melanin. Proteomic analyses identified mainly the accumulation of proteins related to amino acids metabolism, ergosterol synthesis and melanin production, suggesting that P. lutzii responds to copper overload by changing aspects of its metabolism and also plasma membrane and cell wall remodeling. Proteomic data were confirmed by biochemical analysis., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Fungi of the genus Paracoccidioides are the etiological agents of Paracoccidioidomycosis (PCM), the most prevalent mycosis in Latin America. Paracoccidioidomycosis infection is acquired by inhalation of Paracoccidioides conidia, which have first contact with the lungs and can subsequently spread to other organs/tissues. Until now, there have been no proteomic studies focusing on this infectious particle of Paracoccidioides. In order to identify the Paracoccidioides lutzii conidia proteome, conidia were produced and purified. Proteins were characterized by use of the nanoUPLC-MS E approach. The strategy allowed us to identify a total of 242 proteins in P. lutzii conidia. In the conidia proteome, proteins were classified in functional categories such as protein synthesis, energy production, metabolism, cellular defense/virulence processes, as well as other processes that can be important for conidia survival. Through this analysis, a pool of ribosomal proteins was identified, which may be important for the initial processes of dimorphic transition. In addition, molecules related to energetic and metabolic processes were identified, suggesting a possible basal metabolism during this form of resistance of the fungus. In addition, adhesins and virulence factors were identified in the P. lutzii conidia proteome. Our results demonstrate the potential role that these molecules can play during early cell-host interaction processes, as well as the way in which these molecules are involved in environmental survival during this form of propagation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Fungi of the Paracoccidioides genus are the etiological agents of paracoccidioidomycosis (PCM), a systemic mycosis restricted to the countries of Latin America. Currently, the Paracoccidioides complex is represented by Paracoccidioides lutzii , Paracoccidioides americana , Paracoccidioides brasiliensis , Paracoccidioides restrepiensis , and Paracoccidioides venezuelensis . Even with advances in techniques used for diagnosing fungal diseases, high rates of false-positive results for PCM are still presented. Additionally, there is no efficient antigen that can be used to follow up the efficiency of patient treatment. The immunoproteomic is considered a powerful tool for the identification of antigens. In addition, antigens are molecules recognized by the immune system, which make them excellent targets for diagnostic testing of diseases caused by microorganisms. In this vein, we investigated which antigens are secreted by species representing Paracoccidioides complex to increase the spectrum of molecules that could be used for future diagnostic tests, patient follow-up, or PCM therapy. To identify the profile of antigens secreted by Paracoccidioides spp., immunoproteomic approaches were used combining immunoprecipitation, followed by antigen identification by nanoUPLC-MS E -based proteomics. Consequently, it was possible to verify differences in the exoantigen profiles present among the studied species. Through a mass spectrometry approach, it was possible to identify 79 exoantigens in Paracoccidioides species. Using bioinformatics tools, two unique exoantigens in P. lutzii species were identified, as well as 44 epitopes exclusive to the Paracoccidioides complex and 12 unique antigenic sequences that can differentiate between Paracoccidioides species. Therefore, these results demonstrate that Paracoccidioides species have a range of B-cell epitopes exclusive to the complex as well as specific to each Paracoccidioides species. In addition, these analyses allowed us the identification of excellent biomarker candidates for epidemiology screening, diagnosis, patient follow-up, as well as new candidates for PCM therapy., (Copyright © 2020 Moreira, Oliveira, Silva, Inácio, Bailão, Parente-Rocha, Cruz-Leite, Paccez, de Almeida Soares, Weber and Borges.)

Staphylococcus saprophyticus is a Gram-positive and coagulase negative cocci that composes the skin microbiota and can act as an opportunistic agent causing urinary tract infections, being more frequent in sexually active young women. The ability of a pathogen to cause infection in the host is associated to its ability to adhere to host cells and to survive host immune defenses. In this work, we presented the comparative proteomic profile of three S. saprophyticus strains. It was possible to characterize differences in the proteome content, specially related to expression of virulence factors. We compiled this data and previous data and we detected one strain (9325) possessing higher production and secretion of proteins related to virulence. Our results show that phenotypic, genotypic, and proteomic differences reflect in the ability to survive during interaction with host cells, since the 9325 strain presented a higher survival rate after macrophage interaction. In counterpart, the 7108 strain that possesses lower content of proteins related to virulence presented higher ability to form biofilm suggesting that this strain can be better adapted to persist in the host and in the environment. Our work describes, for the first time, proteomic flexibility among S. saprophyticus strains, reflecting in virulence and persistence., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a systemic disorder that involves the lungs and other organs. The adherence of pathogenic microorganisms to host tissues is an essential event in the onset of colonization and spread. The host-pathogen interaction is a complex interplay between the defense mechanisms of the host and the efforts of pathogenic microorganisms to colonize it. Therefore, the identification of fungi proteins interacting with host proteins is an important step understanding the survival strategies of the fungus within the host. In this paper, we used affinity chromatography based on surface proteomics (ACSP) to investigate the interactions of pathogen proteins with host surface molecules. Paracoccidioides lutzii extracts enriched of surface proteins were captured by chromatographic resin, which was immobilized with macrophage cell surface proteins, and identified by mass spectrometry. A total of 215 proteins of P. lutzii were identified interacting with macrophage proteins. In silico analysis classified those proteins according to the presence of sites for N- and O-glycosylation and secretion by classical and non-classical pathways. Serine proteinase (SP) and fructose-1,6-bisphosphate aldolase (FBA) were identified in our proteomics analysis. Immunolocalization assay and flow cytometry both showed an increase in the expression of these two proteins during host-pathogen interaction., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Human adenovirus (HAdV-2) is considered a common agent of respiratory tract infection in the human, especially in children. Virus infection is believed to modify host cell expression necessary for its replication and therefore cell proteome can reflect the changes of specific cellular pathways during infection. This study aims to identify differentially expressed proteins of A549 cells in response to HAdV-2 infection using a label-free liquid chromatography-high-resolution tandem mass spectrometry strategy (LC-MS/MS) at 24 and 48 hpi. A total of 248 and 216 proteins were deregulated by 1.35-fold at 24 and 48 hpi, respectively. Among them, 155 were upregulated at 24 hpi and 86 at 48 hpi, whereas 93 and 130 were downregulated at 24 and 48 hpi, respectively. The identified proteins were involved in different pathways as energy, transcription, protein synthesis, cytoskeleton, rescue and defense, cell cycle, DNA processing, transportation, and metabolism. Glycolytic pathway and histone deregulated proteins were further confirmed by chemical testing and immunofluorescence, respectively. The results suggest that the identified proteins influenced HAdV-2 infection in the context of viral replication and propagation. This study complement proteomic data obtained from previous studies and reinforce the understanding of the relationship between HAdV and host cell., (© 2019 Wiley Periodicals, Inc.)

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MS E technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis . The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite-host relationship and pathogenesis of PCM.

Staphylococcus saprophyticus is a gram-positive microorganism responsible for urinary tract infections (UTIs). Although some virulence factors are characterized, such as urease, autolysins, adhesins and hemagglutinins, large-scale proteomic studies have not been performed within this species. We performed the characterization of the exoproteome from three S. saprophyticus strains: the reference strain ATCC 15,305, a non-capsular strain 7108 and the 9325 strain containing a thick capsule which were cultured in BHI medium and culture supernatants were analysed by using mass spectrometry approach. We observed a core of 72 secreted proteins. In addition, it was possible to detect diversity in the protein profiles of the exoproteomes. Interestingly, strain 7108 presented no secretion of three antigenic proteins, including the classical SsaA antigen. In addition, the level of antigenic proteins secreted by strain 9325 was higher than in ATCC 15,305. This result was confirmed by Western blot analysis using anti-SsaA polyclonal antibodies, and no production/ secretion of SsaA was detected in strain 7108. Transcriptional data shows that 7108 strain produces transcripts encoding SsaA, suggesting post-transcriptional regulation occurs in this strain. Moreover, when compared with the other strains that were analyzed, it was possible to detect higher levels of proteases secreted by strain 7108 and higher levels of antigenic proteins and transglycosylases secreted by 9325 strain. The results reveal diversity in protein secretion among strains. This research is an important first step towards understanding the variability in S. saprophyticus exoproteome profile and could be significant in explaining differences among strains., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

This article presents a proteomic dataset generated from a comparative analysis of the exoproteome of Staphylococcus saprophyticus, ATCC 15305, 7108 and 9325 strains. The extract of secreted proteins were obtained after incubation of stationary phase cells in BHI medium. All samples were submitted to nano-ESI-UPLC-MS E , and the spectrum obtained was processed and analyzed by ProteinLynx Global Server (PLGS), Uniprot and Pedant databases, for identification, annotation and functional classification of proteins. Fold changes and protein relative abundances were properly reported. This report is related to the research article entitled "The exoproteome profiles of three Staphylococcus saprophyticus strains reveal diversity in protein secretion contents" (Oliveira et al., 2018). The proteomic data generated have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with a project number PXD008643, https://www.ebi.ac.uk/pride/archive/projects/PXD008643.

Aim: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action., Methods: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound., Results: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors., Conclusion: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) - an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MS E ) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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.

Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy.

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.

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Paracoccidioidomycosis (PCM) is a systemic granulomatous human mycosis caused by fungi of the genus Paracoccidioides, which is geographically restricted to Latin America. Inhalation of spores, the infectious particles of the fungus, is a common route of infection. The PCM treatment of choice is azoles such as itraconazole, but sulfonamides and amphotericin B are used in some cases despite their toxicity to mammalian cells. The current availability of treatments highlights the need to identify and characterize novel targets for antifungal treatment of PCM as well as the need to search for new antifungal compounds obtained from natural sources or by chemical synthesis. To this end, we evaluated the antifungal activity of a camphene thiosemicarbazide derivative (TSC-C) compound on Paracoccidioides yeast. To determine the response of Paracoccidioides spp. to TSC-C, we analyzed the transcriptional profile of the fungus after 8 h of contact with the compound. The results demonstrate that Paracoccidioides lutzii induced the expression of genes related to metabolism; cell cycle and DNA processing; biogenesis of cellular components; cell transduction/signal; cell rescue, defense and virulence; cellular transport, transport facilities and transport routes; energy; protein synthesis; protein fate; transcription; and other proteins without classification. Additionally, we observed intensely inhibited genes related to protein synthesis. Analysis by fluorescence microscopy and flow cytometry revealed that the compound induced the production of reactive oxygen species. Using an isolate with down-regulated SOD1 gene expression (SOD1-aRNA), we sought to determine the function of this gene in the defense of Paracoccidioides yeast cells against the compound. Mutant cells were more susceptible to TSC-C, demonstrating the importance of this gene in response to the compound. The results presented herein suggest that TSC-C is a promising candidate for PCM treatment.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)