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Paracoccidioidomycosis (PCM) is a chronic endemic mycosis in Latin America, predominantly caused by Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pl01). Diagnosing PCM is challenging due to species-specific antigenic differences, therefore new biomarkers for accurate and rapid detection are needed. This study explores multiple tolerization subtractive immunization (MTSI) to generate monoclonal antibodies against rare or weakly expressed epitopes of Pb18 and Pl01, potentially improving PCM diagnosis. These strains were cultured to obtain cell-free antigens (CFA). MTSI involved immunizing BALB/c mice with CFA from Pb18 as a tolerogen and Pl01 as an immunogen, using Freund's adjuvant and cyclophosphamide to induce immune tolerance. The immune response was monitored via Enzyme-linked immunosorbent assay (ELISA) and Western blotting. Hybridomas were generated by fusing splenocytes from immunized mice with myeloma cells, after which clonal selection was conducted based on reactivity to Pl01 antigens. The study explores the presence of various proteins, including gp43 and Hsp60, in the protein profile of CFAs. Additionally, polyclonal antibody reactivity to Pb18 antigens was significantly reduced, suggesting that MTSI effectively promoted immunological tolerance. Followig the screening of hybridomas, clones with good reactivity to Pl01 and less reactive to Pb18 were selected. The monoclonal clones C1 and E6 exhibited potential specificity for Pl01 antigens. The effective generation of P. lutzii -specific antibodies by MTSI demonstrates this technology's promise for the development of accurate PCM diagnostic instruments. These antibodies have the potential to enhance patient outcomes and reduce the incidence of false-negative diagnoses, which could lead to better disease management.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis., 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 © 2024 Elsevier Ltd. All rights reserved.)

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis , present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro , as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis . PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Medina-Alarcón, Tobias da Silva, Ferin, Pereira-da-Silva, Marcos, dos Santos, Regasini, Chorilli, Mendes-Giannini, Pavan and Fusco-Almeida.)

The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis.

The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and broad-spectrum antifungal activity. Previously, our group demonstrated the promising anti- Paracoccidioides activity of G14. In this work, to evaluate the antifungal characteristics of G14 for Paracoccidioides lutzii , a chemical-genetic interaction analysis was conducted on a Saccharomyces cerevisiae model. N-glycosylation and/or the unfolded protein response pathway was identified as a high-confidence process for drug target prediction. The overactivation of unfolded protein response (UPR) signaling was confirmed using this model with IRE1/ATF6/PERK genes tagged with green fluorescent protein (GFP). In P. lutzii , this prediction was confirmed by the low activity of glycosylated enzymes [α-(1,3)-glucanase, N -acetyl-β-d-glucosaminidase (NAGase), and α-(1,4)-amylase], by hyperexpression of genes involved with the UPR and glycosylated enzymes, and by the reduction in the amounts of glycosylated proteins and chitin. All of these components are involved in fungal cell wall integrity and are dependent on the N-glycosylation process. This loss of integrity was confirmed by the reduction in mitochondrial activity, impaired budding, enhancement of wall permeability, and a decrease in viability. These events led to a reduction of the ability of fungi to adhere on human lung epithelial cells (A549) in vitro Therefore, G14 may have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering with the microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti- Paracoccidioides compound., (Copyright © 2019 American Society for Microbiology.)

The Paracoccidioides brasiliensis strain downregulated the expression of adhesin Pb14-3-3 (Pb14-3-3 aRNA) was evaluated in a murine model of paracoccidioidomycosis (PCM). Pb14-3-3 aRNA displays attenuated virulence and triggered the formation of fewer granulomas by lowering the fungal burden in the lungs. Additionally, the Pb14-3-3 aRNA showed more elongated yeast cells and less ability to induce pneumocytes apoptosis in vitro. Our results show that 14-3-3 is an important virulence factor in P. brasiliensis-induced pulmonary infection., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Paracoccidioidomycosis (PCM) is a fungal disease restricted to Latin countries, and its etiologic agents derive from the Paracoccidioides genus. Attenuation or loss of virulence in Paracoccidioides spp. following successive subculturing has been described. However, virulence can be recovered by passage in mammalian host. In this study, the recovery of adhesion of P. brasiliensis through passage in mice was compared to that in the insect Galleria mellonella. Analysis of in vitro fungal-host cell interaction, gene expression of adhesins, and analysis of the survival curves revealed that Galleria mellonella is useful for the reactivation of P. brasiliensis adhesion.

Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity., (© The Author 2017. 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.)

Paracoccidioides spp., which are temperature-dependent dimorphic fungi, are responsible for the most prevalent human systemic mycosis in Latin America, the paracoccidioidomycosis. The aim of this study was to characterise the involvement of elongation factor Tu (EF-Tu) in Paracoccidioides brasiliensis-host interaction. Adhesive properties were examined using recombinant PbEF-Tu proteins and the respective polyclonal anti-rPbEF-Tu antibody. Immunogold analysis demonstrated the surface location of EF-Tu in P. brasiliensis. Moreover, PbEF-Tu was found to bind to fibronectin and plasminogen by enzyme-linked immunosorbent assay, and it was determined that the binding to plasminogen is at least partly dependent on lysine residues and ionic interactions. To verify the participation of EF-Tu in the interaction of P. brasiliensis with pneumocytes, we blocked the respective protein with an anti-rPbEF-Tu antibody and evaluated the consequences on the interaction index by flow cytometry. During the interaction, we observed a decrease of 2- and 3-fold at 8 and 24 h, respectively, suggesting the contribution of EF-Tu in fungal adhesion/invasion., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis.

Understanding the possible methodologies for the rapid and inexpensive identification of fungal infections is essential for disease diagnosis, but there are some limitations. To help with this problem, serological methods that detect antigens or antibodies are widely used and are useful for the diagnosis of paracoccidioidomycosis (PCM) through the detection of gp43, which is the main antigen employed for the immunodiagnosis of this disease caused by Paracoccidioides brasiliensis. However, the use of gp43 has become restricted because it was recently found that this marker is not identified in the infections caused by Paracoccidioides lutzii. Therefore, it is necessary to identify new antigens in both species or antigens specific for P. lutzii to decrease the morbidity and/or mortality associated with PCM. This review provides a discussion of new diagnostic challenges after the recent discoveries regarding the taxonomy of the Paracoccidioides genus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: 14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model., Results: The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis., Conclusions: Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.

The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Background: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen)., Results: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction., Conclusions: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

Paracoccidioidomycosis is a systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis. Understanding the interactions between P. brasiliensis and the host tissue depends on the study of the different steps of the process of colonization, especially adhesion, in which the pathogen recognizes ligands on the surface of host cells. This study aimed to verify the role of enolase in the host cell-fungus interaction and the ability of enolase to bind to extracellular matrix components, to determine its subcellular localization, and to study the P. brasiliensis enolase amino acid sequence. The data revealed that fibronectin is the major ligand of enolase. Evaluation of the location of enolase at an ultrastructural level revealed that it is distributed in various cellular compartments, but at a high level in the cell wall. The analysis of the amino acid sequence revealed an internal plasminogen-binding motif ((254)FYKADEKKY(262)), which is conserved in most organisms and described as an important interaction site of the enolase with the host cell surface. This suggests that enolase performs additional functions related to the glycolytic pathway and also plays a role of adhesion in P. brasiliensis. Therefore, this study increases the knowledge about the characteristics of enolase and its influence on the binding process of P. brasiliensis., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)