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.

Proteomics of Paracoccidioides lutzii: Overview of Changes Triggered by Nitrogen Catabolite Repression.

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-MSE 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, ß-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.

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis.

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Proteomic Profiling of Paracoccidioides brasiliensis in Response to Phenacylideneoxindol Derivative: Unveiling Molecular Targets and Pathways.

BACKGROUND: The treatment of paracoccidioidomycosis (PCM) is a challenge, and the discovery of new antifungal compounds is crucial. The phenacylideneoxindoles exhibited promising antifungal activity against Paracoccidioides spp., but their mode of action remains unknown. METHODS: Through proteomic analysis, we investigated the effects of (E)-3-(2-oxo-2-phenylethylidene)indolin-2-one on P. brasiliensis. In addition, we investigated the metabolic alterations of P. brasiliensis in response to the compound. Furthermore, the effects of the compound on the membrane, ethanol production, and reactive oxygen species (ROS) production were verified. RESULTS: We identified differentially regulated proteins that revealed significant metabolic reorganization, including an increase in ethanol production, suggesting the activation of alcoholic fermentation and alterations in the rigidity of fungal cell membrane with an increase of the ergosterol content and formation of ROS. CONCLUSIONS: These findings enhance our understanding of the mode of action and response of P. brasiliensis to the investigated promising antifungal compound, emphasizing its potential as a candidate for the treatment of PCM.

Fungal Vaccine Development: State of the Art and Perspectives Using Immunoinformatics.

Fungal infections represent a serious global health problem, causing damage to health and the economy on the scale of millions. Although vaccines are the most effective therapeutic approach used to combat infectious agents, at the moment, no fungal vaccine has been approved for use in humans. However, the scientific community has been working hard to overcome this challenge. In this sense, we aim to describe here an update on the development of fungal vaccines and the progress of methodological and experimental immunotherapies against fungal infections. In addition, advances in immunoinformatic tools are described as an important aid by which to overcome the difficulty of achieving success in fungal vaccine development. In silico approaches are great options for the most important and difficult questions regarding the attainment of an efficient fungal vaccine. Here, we suggest how bioinformatic tools could contribute, considering the main challenges, to an effective fungal vaccine.

Immunoproteomic and immunoinformatic approaches identify secreted antigens and epitopes from Staphylococcus saprophyticus.

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.

Genome-Wide Association Study Reveals CLEC7A and PROM1 as Potential Regulators of Paracoccidioides brasiliensis-Induction of Cytokine Production in Peripheral Blood Mononuclear Cells.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides and the different clinical forms of the disease are associated with the host immune responses. Quantitative trait loci mapping analysis was performed to assess genetic variants associated with mononuclear-cells-derived cytokines induced by P. brasiliensis on 158 individuals. We identified the rs11053595 SNP, which is present in the CLEC7A gene (encodes the Dectin-1 receptor) and the rs62290169 SNP located in the PROM1 gene (encodes CD133) associated with the production of IL-1ß and IL-22, respectively. Functionally, the blockade of the dectin-1 receptor abolished the IL-1ß production in P. brasiliensis-stimulated PBMCs. Moreover, the rs62290169-GG genotype was associated with higher frequency of CD38+ Th1 cells in PBMCs cultured with P. brasiliensis yeasts. Therefore, our research indicates that the CLEC7A and PROM1 genes are important for the cytokine response induced by P. brasiliensis and may influence the Paracoccidioidomycosis disease outcome.

Molecular Interactions of the Copper Chaperone Atx1 of Paracoccidioides brasiliensis with Fungal Proteins Suggest a Crosstalk between Iron and Copper Homeostasis.

Paracoccidioides spp. are endemic fungi from Latin America that cause Paracoccidioidomycosis, a systemic disease. These fungi present systems for high-affinity metal uptake, storage, and mobilization, which counteract host nutritional immunity and mitigate the toxic effects of metals. Regarding Cu mobilization, the metallochaperone Atx1 is regulated according to Cu bioavailability in Paracoccidioides spp., contributing to metal homeostasis. However, additional information in the literature on PbAtx1 is scarce. Therefore, in the present work, we aimed to study the PbAtx1 protein-protein interaction networks. Heterologous expressed PbAtx1 was used in a pull-down assay with Paracoccidioides brasiliensis cytoplasmic extract. Nineteen proteins that interacted with PbAtx1 were identified by HPLC-MSE. Among them, a relevant finding was a Cytochrome b5 (PbCyb5), regulated by Fe bioavailability in Aspergillus fumigatus and highly secreted by P. brasiliensis in Fe deprivation. We validated the interaction between PbAtx1-PbCyb5 through molecular modeling and far-Western analyses. It is known that there is a relationship between Fe homeostasis and Cu homeostasis in organisms. In this sense, would PbAtx1-PbCyb5 interaction be a new metal-sensor system? Would it be supported by the presence/absence of metals? We intend to answer those questions in future works to contribute to the understanding of the strategies employed by Paracoccidioides spp. to overcome host defenses.

Exposure of Paracoccidioides brasiliensis to Mebendazole Leads to Inhibition of Fungal Energy Production.

Paracoccidioidomycosis (PCM) is a fungal disease caused by organisms of the genus Paracoccidioides spp. The treatment of the disease is lengthy and includes several adverse effects. Various methodologies focus on the search for new treatments against fungal disease, including the repositioning of drugs. Our group showed the fungicidal effect of mebendazole in P. brasiliensis cells. Thus, understanding the effect of exposing fungal cells to mebendazole is significant for further studies in order to demonstrate it as a potential drug for the treatment of PCM. A proteomic analysis of P. brasiliensis exposed to mebendazole was carried out. Analyses showed that exposure strongly affected the pathways related to energy production, such as glycolysis, fermentation, and the electron transport chain. The quantification of adenosine triphosphate (ATP) and mitochondrial activity demonstrated that the drug alters the electron chain, resulting in an increase in oxidative stress. Enzymes such as superoxide dismutase (SOD) and cytochrome c oxidase (Cyt C) were repressed in cells exposed to mebendazole. The concentration of ethanol produced by the cells under treatment demonstrated that the attempt to produce energy through fermentation is also arrested. Thus, the drug inhibits fungal growth through changes in energy metabolism, making it a promising compound for use in the treatment of PCM.

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.

Paracoccidioides brasiliensis plasma membrane characterization by EPR spectroscopy and interactions with amphotericin B, miltefosine and nerolidol.

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to characterize the interactions of amphotericin B (AmB), miltefosine (MIL) and nerolidol (NER) with the plasma membrane of Paracoccidioides brasiliensis. Spin-labeled analogs of stearic acid and steroid androstane distributed into the plasma membrane of the fungus treated with AmB, showed strong interactions with putative AmB/sterol complexes. The observed increase in the EPR parameter 2A// caused by AmB can be interpreted as a remarkable reduction in the spin label mobility and/or an increase in the local polarity. The 2A// parameter reduced gradually as the concentration of MIL and NER increased. The membrane-water partition coefficient (KM/W) of the three compounds under study was estimated based on the minimum concentration of the compounds that causes a change in EPR spectrum. The KM/W values indicated that the affinity of the compounds for the P. brasiliensis membrane follows the order: AmB > MIL > NER. The minimum inhibitory concentration (MIC) values were lower than the respective minimum concentrations of the compounds to cause a change in the EPR spectrum, being ∼3.5-fold lower for AmB, 3.9-fold for MIL and ∼1.4-fold for NER. Taken together, the EPR spectroscopy results suggest that the anti-proliferative effects of the three compounds studied are associated with alterations in cell membranes. One of the most likely consequences of these changes would be electrolyte leakage.Communicated by Ramaswamy H. Sarma.

Paracoccidioides lutzii Formamidase Contributes to Fungal Survival in Macrophages.

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.

Iron Deprivation Modulates the Exoproteome in Paracoccidioides brasiliensis.

Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe3+ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MSE. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.

A structure-based approach for the discovery of inhibitors against methylcitrate synthase of Paracoccidioides lutzii.

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in Latin America, caused by fungi of the genus Paracoccidioides. The treatment of PCM is complex, requiring a long treatment period, which often results in serious side effects. The aim of this study was to screen for inhibitors of a specific target of the fungus that is absent in humans. Methylcitrate synthase (MCS) is a unique enzyme of microorganisms and is responsible for the synthesis of methylcitrate at the beginning of the propionate degradation pathway. This pathway is essential for several microorganisms, since the accumulation of propionyl-CoA can impair virulence and prevent the development of the pathogen. We performed the modeling and molecular dynamics of the structure of Paracoccidioides lutzii MCS (PlMCS) and performed a virtual screening on 89,415 compounds against the active site of the enzyme. The compounds were selected according to the affinity and efficiency criteria of in vitro tests. Six compounds were able to inhibit the enzymatic activity of recombinant PlMCS but only the compound ZINC08964784 showed fungistatic and fungicidal activity against Paracoccidioides spp. cells. The analysis of the interaction profile of this compound with PlMCS showed its effectiveness in terms of specificity and stability when compared to the substrate (propionyl-CoA) of the enzyme. In addition, this compound did not show cytotoxicity in mammalian cells, with an excellent selectivity index. Our results suggest that the compound ZINC08964784 may become a promising alternative antifungal against Paracoccidioides spp. Communicated by Ramaswamy H. Sarma.

Proteomic Response of Paracoccidioides brasiliensis Exposed to the Antifungal 4-Methoxynaphthalene-N-acylhydrazone Reveals Alteration in Metabolism.

Background: Paracoccidioidomycosis is a neglected mycosis with a high socioeconomic impact that requires long-term treatment with antifungals that have limitations in their use. The development of antifungals targeting essential proteins that are present exclusively in the fungus points to a potentially promising treatment. Methods: The inhibitor of the enzyme homoserine dehydrogenase drove the synthesis of N'-(2-hydroxybenzylidene)-4-methoxy-1-naphthohydrazide (AOS). This compound was evaluated for its antifungal activity in different species of Paracoccidioides and the consequent alteration in the proteomic profile of Paracoccidioides brasiliensis. Results: The compound showed a minimal inhibitory concentration ranging from 0.75 to 6.9 µM with a fungicidal effect on Paracoccidioides spp. and high selectivity index. AOS differentially regulated proteins related to glycolysis, TCA, the glyoxylate cycle, the urea cycle and amino acid metabolism, including homoserine dehydrogenase. In addition, P. brasiliensis inhibited protein synthesis and stimulated reactive oxygen species in the presence of AOS. Conclusions: AOS is a promising antifungal agent for the treatment of PCM, targeting important metabolic processes of the fungus.

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.

Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples.

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.