The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

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.

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.

Proteomic alterations in Paracoccidioides brasiliensis caused by exposure to curcumin.

Paracoccidioides spp. are the etiological agent of paracoccidioidomycosis, a disease that causes skin lesions and affect the lungs and other organs. The current management of the disease is long and has several side effects that often lead the patient to give up the treatment, sequelae and even death. The search for new forms of treatment that minimize these drawbacks is very important. Thus, natural compounds are targets of great interest. Curcumin is one of the main components of the tubers of Curcuma longa, presenting medicinal effects well described in the literature, including the antifungal effect on Paracocidioides brasiliensis. Nevertheless, the mechanisms related to the antifungal effect of such compound are still unknown, so the objective of the present research is to understand what changes occur in the metabolism of P. brasiliensis after exposure to curcumin and to identify the main targets of the compound. Proteomic analysis as based on nanoUPLC-MS analysis and the functional classification of the identified proteins. The main metabolic processes that were being regulated were biologically validated through assays such as fluorescence microscopy, EPR and phagocytosis. Proteomic analysis revealed that curcumin regulates several metabolic processes of the fungus, including important pathways for energy production, such as the glycolytic pathway, beta oxidation and the glyoxylate cycle. Protein synthesis was down-regulated in fungi exposed to curcumin. The electron transport chain and the tricarboxylic acid cycle were also down-regulated, indicating that both the mitochondrial membrane and the mitochondrial activity were compromised. Plasma membrane and cell wall structure were altered following exposure to the compound. The fungus' ability to survive the phagocytosis process by alveolar macrophages was reduced. Thus, curcumin interferes with several metabolic pathways in the fungus that causes paracoccidioidomycosis. BIOLOGICAL SIGNIFICANCE: The challenges presented by the current treatment of paracoccidioidomycosis often contributing to patients' withdrawal from treatment, leading to sequelae or even death. Thus, the search for new treatment options against this disease is growing. The discovery that curcumin is active against Paracoccidioides was previously reported by our study group. Here, we clarify how the compound acts on the fungus causing its growth inhibition and decreased viability. Understanding the mechanisms of action of curcumin on P. brasiliensis elucidates how we can seek new alternatives and which metabolic pathways and molecular targets we should focus on in this incessant search to bring the patient a treatment with fewer adverse effects.

In vitro and in silico analysis reveals antifungal activity and potential targets of curcumin on Paracoccidioides spp.

The search for new compounds with activity against Paracoccidioides, etiologic agents of Paracoccidioidomycosis (PCM), is extremely necessary due to the current scenario of the available therapeutic arsenal. Treatment is restricted to three classes of antifungals with side effects. Curcumin is a polyphenol with antifungal effects that is extracted from Curcuma longa. The present work aimed to evaluate the activity of curcumin in different species of Paracoccidioides and to evaluate the potential molecular targets of curcumin using computational strategies. In addition, interactions with classic antifungals used in the treatment of PCM were evaluated. Curcumin inhibits the growth of Paracoccidioides spp. exerting a fungicidal effect. The combination of curcumin with amphotericin B, co-trimoxazole, and itraconazole showed a synergistic or additive interaction. Molecular targets as superoxide dismutase, catalase, and isocitrate lyase were proposed based on in silico approaches. Curcumin affects the fungal plasma membrane and increases the production of reactive oxygen species. Therefore, curcumin is a good alternative for the treatment of PCM.

Overview of Antifungal Drugs against Paracoccidioidomycosis: How Do We Start, Where Are We, and Where Are We Going?

Paracoccidioidomycosis is a neglected disease that causes economic and social impacts, mainly affecting people of certain social segments, such as rural workers. The limitations of antifungals, such as toxicity, drug interactions, restricted routes of administration, and the reduced bioavailability in target tissues, have become evident in clinical settings. These factors, added to the fact that Paracoccidioidomycosis (PCM) therapy is a long process, lasting from months to years, emphasize the need for the research and development of new molecules. Researchers have concentrated efforts on the identification of new compounds using numerous tools and targeting important proteins from Paracoccidioides, with the emphasis on enzymatic pathways absent in humans. This review aims to discuss the aspects related to the identification of compounds, methodologies, and perspectives when proposing new antifungal agents against PCM.

Profile of Enterobacteria Resistant to Beta-Lactams.

A serious emerging problem worldwide is increased antimicrobial resistance. Acquisition of coding genes for evasion methods of antimicrobial drug mechanisms characterizes acquired resistance. This phenomenon has been observed in Enterobacteriaceae family. Treatment for bacterial infections is performed with antibiotics, of which the most used are beta-lactams. The aim of this study was to correlate antimicrobial resistance profiles in Enterobacteriaceae by phenotypic methods and molecular identification of 14 beta-lactamase coding genes. In this study, 70 exclusive isolates from Brazil were used, half of which were collected in veterinary clinics or hospitals Phenotypic methodologies were used and real-time PCR was the molecular methodology used, through the Sybr Green system. Regargding the results found in the tests it was observed that 74.28% were resistant to ampicillin, 62.85% were resistant to amoxicillin associated with clavalunate. The mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of species were blaGIM and blaSIM (66.66% of the samples) and the one that was amplified in a smaller number of samples was blaVIM (16.66%). Therefore, high and worrying levels of antimicrobial resistance have been found in enterobacteria, and a way to minimize the accelerated emergence of their resistance includes developing or improving techniques that generate diagnoses with high efficiency and speed.