Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis induces the death of mouse lymphocytes in a mechanism dependent on Toll-like receptor 4 and tumor necrosis factor.

Paracoccidioides fungi are thermodimorphic microorganisms that cause paracoccidioidomycosis (PCM), an autochthonous disease from Latin America, with most cases in Brazil. Humans become infected by inhaling conidia or mycelial fragments that transform into yeast at body temperature. These fungi cause chronic-granulomatous inflammation, which may promote fibrosis and parenchyma destruction in the lungs. In response to stress imposed by the host, fungi Paracoccidioides spp. increase the expression of heat shock proteins (HSP), which protect them by sustaining cellular proteostasis. Our group has studied the role of HSP60 in PCM, and previous data show that the recombinant HSP60 (rHSP60) has a deleterious effect when used in a single dose as therapy for experimental PCM. Here, we investigated the mechanism by which rHSP60 could worsen the disease. We found that rHSP60 caused the viability loss of splenic or lymph node cells from both immunized and non-immunized mice, including in splenic T lymphocytes under polyclonal stimulation with concanavalin A, probably by undergoing apoptosis. Among analyzed splenic cells, lymphocytes were indeed the main cells to die. When we investigated the death mechanisms, remarkably, we found that there was no viability loss in rHSP60-stimulated splenic cells from mice deficient in Toll-like receptor 4, TRIF adapter protein, and TNF receptor 1(TNFR1), as well as rHSP60-stimulated WT cells incubated with anti-TNF antibody. Besides, caspase-8 inhibitor IETD-CHO blocked the rHSP60 effect on splenic cells, suggesting that rHSP60 induces the extrinsic apoptosis pathway dependent on signaling via TLR4/TRIF and TNFR1.

Extracellular vesicles from virulent P. brasiliensis induce TLR4 and dectin-1 expression in innate cells and promote enhanced Th1/Th17 response.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that transport several biomolecules and are involved in important mechanisms and functions related to the pathophysiology of fungal diseases. EVs from Paracoccidioides brasiliensis, the main causative agent of Paracoccidioidomycosis (PCM), modulate the immune response of macrophages. In this study, we assessed the EVs proteome from a virulent P. brasiliensis isolated from granulomatous lesions and compared their immunomodulatory ability with EVs isolated from the fungus before the animal passage (control EVs) when challenging macrophages and dendritic cells (DCs). Proteome showed that virulent EVs have a higher abundance of virulence factors such as GP43, protein 14-3-3, GAPDH, as well as virulence factors never described in PCM, such as aspartyl aminopeptidase and a SidJ analogue compared with control EVs. Virulent extracellular vesicles induced higher expression of TLR4 and Dectin-1 than control EVs in macrophages and dendritic cells (DCs). In opposition, a lower TLR2 expression was induced by virulent EVs. Additionally, virulent EVs induced lower expression of CD80, CD86 and TNF-α, but promoted a higher expression of IL-6 and IL-10, suggesting that EVs isolated from virulent P. brasiliensis-yeast promote a milder DCs and macrophage maturation. Herein, we showed that EVs from virulent fungi stimulated a higher frequency of Th1/Tc1, Th17, and Treg cells, which gives new insights into fungal extracellular vesicles. Taken together, our results suggest that P. brasiliensis utilizes its EVs as virulence bags that manipulate the immune system in its favour, creating a milder immune response and helping with fungal evasion from the immune system.

Molecular detection of Paracoccidioides spp. in environmental samples from the Southwestern Amazon.

Paracoccidioidomycosis is an infection with the potential for environmental dissemination, especially in regions of hot and humid climate, where human cases have been recorded in the Southwestern Amazon of Brazil, specifically in the state of Acre. Despite studies providing information about the presence of these fungi in soil and animal samples, such as armadillos, further investigations are still needed to determine the epidemiological distribution of the genus Paracoccidioides. The aim of this study was to detect the occurrence of Paracoccidioides fungi in the Southwestern Amazon. To achieve this, 60 soil samples were collected from armadillo burrows on rural properties in the in the municipalities of Acrelândia, Bujari, Plácido de Castro, Rio Branco, Sena Madureira, and Senador Guiomard, located in the state of Acre, Brazil. Fungal DNA was extracted from these samples using the DNEASY® PowerSoil kit-Quiagen, followed by Nested PCR technique with ITS4 and ITS5 as external primers, and PBITS-E and PBITS-R as internal primers. DNA amplification products of about 380 bp compatible with Paracoccidioides spp. were detected in six samples (10%), being sequenced and identified as P. brasiliensis. These findings indicate that the soils of the Acre state could be considered a potential source for Paracoccidioides spp., suggesting that local infections are likely.

Structure of glyceraldehyde-3-phosphate dehydrogenase from Paracoccidioides lutzii in complex with an aldonic sugar acid.

The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of 'GAA site'. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose.

Synthesis of 1,3,4-oxadiazoles with 4-methoxynaphthalene ring: discovering new compounds with antimicrobial activity.

Background: Paracoccidioidomycosis (PCM) is a systemic infection caused by Paracoccidioides spp. (Pb). PCM can be associated or clinically confused with tuberculosis (TB), another pulmonary infection, caused by Mycobacterium tuberculosis (Mtb). Futhermore, the long treatment time of TB and PCM and the cases of TB drug resistance impose difficulties for the cure of these diseases. Results: New 1,3,4-oxadiazoles containing the 4-methoxynaphthalene ring were synthesized and their antimicrobial activity was evaluated against Pb and Mtb. The derivative 6n (with 2-hydroxy-5-nitrophenyl subunit) is the most promising of the series. Conclusion: The 1,3,4-oxadiazole 6n can be used as a prototype drug candidate, with anti-Pb and anti-MTb activities, showing a broad-spectrum profile for the treatment of both pulmonary infections.

Proteomic identification of metabolic changes in Paracoccidioides brasiliensis induced by a nitroheteroarylchalcone.

Aim: To access the metabolic changes caused by a chalcone derivative (LabMol-75) through a proteomic approach. Methods: Proteomic analysis was performed after 9 h of Paracoccidioides brasiliensis yeast (Pb18) cell incubation with the LabMol-75 at MIC. The proteomic findings were validated through in vitro and in silico assays. Results: Exposure to the compound led to the downregulation of proteins associated with glycolysis and gluconeogenesis, ß-oxidation, the citrate cycle and the electron transport chain. Conclusion: LabMol-75 caused an energetic imbalance in the fungus metabolism and deep oxidative stress. Additionally, the in silico molecular docking approach pointed to this molecule as a putative competitive inhibitor of DHPS.

A taxonomic review of the genus Paracoccidioides, with focus on the uncultivable species.

Paracoccidioides species have always been surrounded by taxonomic uncertainties. The continuing nomenclatoral muddle was caused in part by the failure of Adolfo Lutz and Jorge Lôbo to name the etiologic agents of human paracoccidioidomycosis and Jorge Lôbo's diseases, respectively. Early in their history, it was postulated that the cultivable species causing systemic infections belonged in the genus Paracoccidioides, whereas the uncultivable species, causing skin disease, were not part of the genus. The taxonomy of these pathogens was further complicated when a similar skin disease with numerous yeast-like cells in infected dolphins was also reported. Due to its phenotypic similarities with that described by Jorge Lôbo in human and its uncultivable nature, it was assumed that the disease in dolphins was caused by the same fungus. Recent molecular and population genetic analysis, however, found the DNA extracted from the uncultivable yeast-like cells affecting dolphins shared common phylogenetic traits with cultivable Paracoccidioides species. The study revealed that the uncultivable pathogens comprised 2 different Paracoccidioides species, now known as P. ceti and P. loboi, correspondingly. To validate P. loboi binomial, a comprehensive historical critical review of Jorge Lôbo etiology was performed. This review showed the proposed binomial P. loboi was previously used, and, thus, a replacement name is introduced, Paracoccidioides lobogeorgii nom. nov. In addition, in this review, several cultivable human Paracoccidioides species are validated, and the generic type species, P. brasiliensis, is neotypified as the original material could not be traced.

The story of Paracoccidiodes gp43.

This review is about Dr. Luiz Rodolpho Raja Gabaglia Travassos' scientific contributions to paracoccidioidomycosis as told by myself, Rosana Puccia, but co-written with Dr. Carlos P. Taborda, my younger scientific brother, collaborator, and dear friend. Dr. Travassos' pioneer papers and scientific insights covering biochemistry, immunology, cell biology, and molecular biology in the paracoccidiodomycosis area are key contributions that we acknowledge here, with focus on the Paracoccidioides antigen gp43. Importantly, we tell some personal stories behind the scene. Dr. Travassos' contribution to science is available in a number of quality publications, while his influence to hundreds of people who gravitated around him will be kept alive inside each one of us forever.

Itraconazole and neutrophil interactions in the immune-inflammatory response of paracoccidioidomycosis using a murine air pouch infection model.

Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis (Pb), is a severe mycosis, prevalent in tropical countries. The presence of polymorphonuclear neutrophils (PMN) in lesions is conspicuous, indicating their central role in innate immunity through the direct killing of Pb and the production of cytokines that activate acquired immunity in the presence of itraconazole (Itra). The toxicity and direct antifungal activity of Itra on Pb in splenocyte co-cultures were evaluated in vitro. Itra showed no toxic effect but marked antifungal activity against Pb. Purified PMN were obtained by the subcutaneous (SC) injection of Pb into mice. Results showed the effect of Itra on the size of the air pouch produced, the cellular population that migrated to the infection site, protein, and mitochondrial metabolism patterns, production of ROS an NO, and the number of cytokines synthesized. Lower doses (3 and 10 mg/kg) of Itra did not affect the cellular profile but led to a lower influx of viable more active PMN, and increased production of ROS and proteins. At a dose of 50 mg/kg the PMN profile remained unchanged along with all other parameters analyzed remained unaltered. Decreases in most cytokine levels were inversely proportional to the Itra concentration. Lower Itra concentrations may elicit activation of the immune response because the combined effects of therapy and immune response are needed, while the higher dose does not require it. Itra also promotes the activation of the cytokines which elicit PMN activation and consequently the resolution of Pb18 infection in the air pouch.

Acute paracoccidioidomycosis worsened by immunosuppressive therapy due to a misdiagnosis of Crohn's disease.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, mostly in Brazil. The involvement of the gastrointestinal tract is uncommon and usually associated with the acute form. Recently, a cluster of acute PCM cases has been described in Rio de Janeiro, Brazil. We report a 42-year-old male, resident of Rio de Janeiro, presenting chronic diarrhea and abdominal pain in the past 3 years, previously diagnosed as Chron´s disease. When immunosuppressive therapy was prescribed, the patient evolved with worsening of the previous symptoms in addition to odynophagia, 20 kg-weight loss, disseminated skin lesions, diffuse lymphadenopathy and adrenal insufficiency. Histopathological and mycological examination of a skin lesion were compatible with PCM. Itraconazole was prescribed in high doses (400 mg/day). After seven months of treatment, the patient presented with acute abdominal pain which led to an emergent appendectomy, revealing the presence of the fungus. After 24 months, the patient reached clinical cure and recovered from adrenal insufficiency. We emphasize the importance of PCM as a differential diagnosis in patients with chronic diarrhea. The risk of fungal infections should be considered prior to initiating immunosupressive therapies, particularly in endemic areas.

Role of paracoccin on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs in the Galleria mellonella larvae model.

Paracoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN's dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs' N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes' fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes.

Chronic exposure to cigarette smoke transiently worsens the disease course in a mouse model of pulmonary paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) may present as an acute/subacute clinical form, characterized by a progressive disease arising from the airborne initial infection, or, most often, as an asymptomatic or subclinical infection that may manifest later during an individual's life, the chronic form. Epidemiological studies show the existence of a strong association between smoking and the development of the chronic form. Current evidence demonstrates that cigarette smoke (CS) has immunosuppressive properties that could be implicated in the increasing susceptibility to the chronic form of PCM. To address this issue, we developed a murine model of a non-progressive pulmonary form of PCM that was exposed to CS at a magnitude that mimicked a moderate smoker. The chronic CS exposure started after 2 weeks and lasted up until 20 weeks post-infection, with the aim of mimicking human natural history, since it is estimated that individuals from endemic areas are infected early in life. The control group consisted of infected but not CS-exposed mice. We assessed the lung fungal burden (colony forming units [CFU]) and the area affected by the granulomatous inflammatory response, fungal dissemination to spleen and liver, and, by immunohistochemistry, the presence of CD4 and CD8 lymphocytes, CD68 and MAC-2 macrophages, and IFN-γ, IL-10 and TNF expressing cells within the granulomatous response. We detected a CS effect as early as 2 weeks after exposure (four weeks post-infection) when the lung CFU of exposed animals was significantly higher than in their non-exposed counterparts. At 12 weeks, the CS-exposed animals presented a more severe disease, as witnessed by the persistent higher lung fungal load (although it did not reach statistical significance [ p = 0.054]), greater dissemination to other organs, greater affected area of the lung, decreased IFN-γ/IL-10 ratio, and higher TNF expression within the granulomas, compared with CS-non-exposed mice. The number of CD4 and CD8 lymphocytes infiltrating the granulomas was similar between both mice groups, but there was a decrease in the number of MAC-2+ macrophages. No difference was noted in the CD68+ macrophage number. However, the follow-up in week 20 showed that the immunological effects of exposure to CS ceased, with both CS and NCS mice showing the same infectious features, i.e., a trend for resolution of the infection. In conclusion, we show that chronic CS-exposure alters the course of the disease in an experimental model of subclinical pulmonary PCM, confirming the epidemiological link between CS-exposure and the chronic form of PCM. However, we also show that this effect is transitory, being detected between 4- and 12-weeks post-infection but not thereafter. The possible immune mechanisms that mediate this effect and the reasons for its transitory effect are discussed.

The Impact of Paracoccidioides spp Infection on Central Nervous System Cell Junctional Complexes.

Paracoccidioidomycosis (PCM), a systemic mycosis caused by the fungus Paracoccidioides spp. is the most prevalent fungal infection among immunocompetent patients in Latin America. The estimated frequency of central nervous system (CNS) involvement among the human immunodeficiency virus (HIV)/PCM-positive population is 2.5%. We aimed to address the impact of neuroparacoccidioidomycosis (NPCM) and HIV/NPCM co-infection on the tight junctions (TJ) and adherens junction (AJ) proteins of the CNS. Four CNS formalin-fixed paraffin-embedded (FFPE) tissue specimens were studied: NPCM, NPCM/HIV co-infection, HIV-positive without opportunistic CNS infection, and normal brain autopsy (negative control). Immunohistochemistry was used to analyze the endothelial cells and astrocytes expressions of TJ markers: claudins (CLDN)-1, -3, -5 and occludin; AJ markers: ß-catenin and E-cadherin; and pericyte marker: alpha-smooth muscle actin. FFPE CNS tissue specimens were analyzed using the immunoperoxidase assay. CLDN-5 expression in the capillaries of the HIV/NPCM coinfected tissues (mixed clinical form of PCM) was lower than that in the capillaries of the HIV or NPCM monoinfected (chronic clinical form of PCM) tissues. A marked decrease in CLDN-5 expression and a compensatory increase in CLDN-1 expression in the NPCM/HIV co-infection tissue samples was observed. The authors suggest that Paracoccidioides spp. crosses the blood-brain barrier through paracellular pathway, owing to the alteration in the CLDN expression, or inside the macrophages (Trojan horse).

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.

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.

Compulsory notification of paracoccidioidomycosis: A 14-year retrospective study of the disease in the state of Paraná, Brazil.

BACKGROUND: Paracoccidioidomycosis, caused by fungi of the Paracoccidioides genus, is one of the most important endemic mycoses in Brazil. The disease is not of mandatory reporting in the country; however, some Brazilian states, such as Paraná, have included it on their local lists of public health-reportable diseases. OBJECTIVE: Describe the epidemiology of the positive paracoccidioidomycosis cases in the state of Paraná based on analysis of reporting forms and mortality data. SUBJECTS AND METHODS: Data of positive cases of state residents 2007-2020 were obtained from public health databases and frequency, incidence, geographic distribution, mortality and trends were analysed. Mortality of the disease was also compared to other mycoses such as cryptococcosis, aspergillosis, histoplasmosis, candidiasis and sporotrichosis. RESULTS: 670 patients were positive for the disease. The cumulative and average annual incidence was, respectively, 6.4 and 0.46 cases/100,000 inhabitants. The new cases of paracoccidioidomycosis were reported mainly by specialised health units, including tertiary centres, and 285 days was the mean from the beginning of the symptoms until the diagnosis. The western region showed the highest incidence and mortality by the disease over the other state mesoregions. During the period, a decreasing trend was observed in the confirmed cases and stability in the mortality rate with an average annual mortality of 1.17 per million/inhabitants in the state; however, paracoccidioidomycosis had the highest mortality when compared to other mycoses. CONCLUSIONS: Paracoccidioidomycosis is an important endemic mycosis in Paraná and this study provides an epidemiological baseline for future modifications of paracoccidioidomycosis surveillance.

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.

Antibody- Based Immunotherapy Combined With Antimycotic Drug TMP- SMX to Treat Infection With Paracoccidioides brasiliensis.

Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of Paracoccidioides spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall ß-glucan polymer. In vitro, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts. In vivo, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- Î³, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.

Molecular epidemiology of Paracoccidiodes spp. recovered from patients with paracoccidioidomycosis in a teaching hospital from Minas Gerais State of Brazil.

INTRODUCTION: Paracoccidioidomycosis (PCM) is caused by several species of the Paracoccidioides genus which can be differentiated by interspecific genetic variations, morphology and geographic distribution. Intraspecific variability correlation with clinical and epidemiological aspects of these species still remains unclear. This study aimed to sequence the loci GP43, exon 2 and ARF of 23 clinical isolates of Paracoccidioides spp. from patients in the Southeast Region of Brazil. METHODOLOGY AND MAIN FINDINGS: GenBank was used to compare the present (23) with previous described sequences (151) that included ARF and GP43. It was identified a high polymorphism rate among the 23 isolates in comparison to the other 151. Among the isolates, 22 (95.66%) were S1/P. brasiliensis and 1 (4.34%) was identified as PS2/P. americana. A total of 45 haplotypes were found as follows: 19 from S1/P. brasiliensis (13 from the present study), 15 from P. lutzii, 6 from PS2/P. americana (1 from the present study), 3 from PS3/P. restrepiensis and 2 from PS4/P. venezuelensis. Moreover, exclusive haplotypes according to clinical origin and geographical area were found. S1/P. brasiliensis (HD = 0.655 and K = 4.613) and P. lutzii (HD = 0.649 and K = 2.906) presented the highest rate of polymorphism among all species, from which 12 isolates of the present study were clustered within S1b/P. brasiliensis. The GP43 locus showed a higher variability and was found to be the main reason for the species differentiation. CONCLUSIONS: The results herein decribed show a high intraspecific genetic variability among S1/P. brasiliensis isolates and confirm the predominance of this species in the Southeast region of Brazil. The finding of exclusive haplotypes according to clinical origin and geographical area would suggest correlation between the molecular profile with the clinical form and geographic origin of patients with PCM.

The taxonomy of two uncultivated fungal mammalian pathogens is revealed through phylogeny and population genetic analyses.

Ever since the uncultivated South American fungal pathogen Lacazia loboi was first described 90 years ago, its etiology and evolutionary traits have been at the center of endless controversies. This pathogen infects the skin of humans and as long believed, dolphin skin. However, recent DNA analyses of infected dolphins placed its DNA sequences within Paracoccidioides species. This came as a surprise and suggested the human and dolphin pathogens may be different species. In this study, population genetic analyses of DNA from four infected dolphins grouped this pathogen in a monophyletic cluster sister to P. americana and to the other Paracoccidioides species. Based on the results we have emended the taxonomy of the dolphin pathogen as Paracoccidioides cetii and P. loboi the one infecting human. Our data warn that phylogenetic analysis of available taxa without the inclusion of unusual members may provide incomplete information for the accurate classification of anomalous species.