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22 pages, 4326 KiB

Open AccessArticle

Drk1, a Dimorphism Histidine Kinase, Contributes to Morphology, Virulence, and Stress Adaptation in Paracoccidioides brasiliensis

by Caroline Maria Marcos, Haroldo Cesar de Oliveira, Patrícia Akemi Assato, Rafael Fernando Castelli, Ana Marisa Fusco-Almeida and Maria José Soares Mendes-Giannini

J. Fungi 2021, 7(10), 852; https://doi.org/10.3390/jof7100852 - 12 Oct 2021

Cited by 1 | Viewed by 2137

Abstract

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity [...] Read more.

P. brasiliensis is a thermally dimorphic fungus belonging to Paracoccidioides complex, causative of a systemic, endemic mycosis limited to Latin American countries. Signal transduction pathways related to important aspects as surviving, proliferation according to the biological niches are linked to the fungal pathogenicity in many species, but its elucidation in P. brasiliensis remains poorly explored. As Drk1, a hybrid histidine kinase, plays regulators functions in other dimorphic fungi species, mainly in dimorphism and virulence, here we investigated its importance in P. brasilensis. We, therefore generated the respective recombinant protein, anti-PbDrk1 polyclonal antibody and a silenced strain. The Drk1 protein shows a random distribution including cell wall location that change its pattern during osmotic stress condition; moreover the P. brasiliensis treatment with anti-PbDrk1 antibody, which does not modify the fungus’s viability, resulted in decreased virulence in G. mellonella model and reduced interaction with pneumocytes. Down-regulating PbDRK1 yielded phenotypic alterations such as yeast cells with more elongated morphology, virulence attenuation in G. mellonella infection model, lower amount of chitin content, increased resistance to osmotic and cell wall stresses, and also caspofungin, and finally increased sensitivity to itraconazole. These observations highlight the importance of PbDrk1 to P. brasiliensis virulence, stress adaptation, morphology, and cell wall organization, and therefore it an interesting target that could help develop new antifungals. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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16 pages, 2660 KiB

Open AccessArticle

Monocyte-Derived Dendritic Cells Can Revert In Vitro Antigen-Specific Cellular Anergy in Active Human Paracoccidioidomycosis

by Paula Keiko Sato, Telma Miyuki Oshiro, Érika Cano Passos, Tatiana Giselle Rodrigues Miranda, Constância Lima Diogo, Claudia de Abreu Fonseca, Aya Sadahiro, Sandro Rogério de Almeida and Maria Aparecida Shikanai-Yasuda

J. Fungi 2021, 7(3), 201; https://doi.org/10.3390/jof7030201 - 10 Mar 2021

Cited by 1 | Viewed by 2087

Abstract

We investigated the in vitro effects of two Paracoccidioides brasiliensis antigens on monocyte-derived dendritic cells (moDCs) from patients with paracoccidioidomycosis (PCM). MoDCs from patients with active or treated PCM and non-PCM subjects were generated, stimulated with TNF-α, and P. brasiliensis antigens, 43 kDa [...] Read more.

We investigated the in vitro effects of two Paracoccidioides brasiliensis antigens on monocyte-derived dendritic cells (moDCs) from patients with paracoccidioidomycosis (PCM). MoDCs from patients with active or treated PCM and non-PCM subjects were generated, stimulated with TNF-α, and P. brasiliensis antigens, 43 kDa glycoprotein (gp43) and cell-free antigen (CFA), and analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISA). Our data revealed that patients with PCM had a high frequency of HLA-DR⁺ cells, but the treated group had more CD86⁺ cells with increased IL-12p40. Patients with active PCM had more CD80⁺ moDCs, and as a novel finding, large amounts of chemokine (C-C motif) ligand 18 (CCL18) in the supernatants from their in vitro moDC cultures. Both gp43- and CFA-stimulated moDCs from the patients with PCM successfully reverted the in vitro antigen-specific anergy, inducing a proliferative response. However, CFA-stimulated moDCs led to higher lymphoproliferation, with increased IFN-γ and TNF-α in the cells from the patients with active PCM compared with gp43. These original results combined with constant IL-10 and increased IL-12p40 levels suggest that a more complex antigen, such as CFA, may be a better inducer of the protective Th1 immune response than purified gp43 is, and a suitable target for future studies on anti-P. brasiliensis dendritic cell (DC)-based vaccines. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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20 pages, 3052 KiB

Open AccessArticle

A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species

by Breno Gonçalves Pinheiro, Ana Paula Pôssa, Paula Portella Della Terra, Jamile Ambrósio de Carvalho, Giannina Ricci, Angela Satie Nishikaku, Rosane Christine Hahn, Zoilo Pires de Camargo and Anderson Messias Rodrigues

J. Fungi 2021, 7(3), 169; https://doi.org/10.3390/jof7030169 - 26 Feb 2021

Cited by 13 | Viewed by 3890

Abstract

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent [...] Read more.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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23 pages, 3348 KiB

Open AccessArticle

Differential miRNA Expression in Human Macrophage-Like Cells Infected with Histoplasma capsulatum Yeasts Cultured in Planktonic and Biofilm Forms

by Nayla de Souza Pitangui, Junya de Lacorte Singulani, Janaina de Cássia Orlandi Sardi, Paula Carolina de Souza, Gabriela Rodríguez-Arellanes, Blanca Estela García-Pérez, Francisco Javier Enguita, Fernando R. Pavan, Maria Lucia Taylor, Maria José Soares Mendes-Giannini and Ana Marisa Fusco-Almeida

J. Fungi 2021, 7(1), 60; https://doi.org/10.3390/jof7010060 - 18 Jan 2021

Cited by 2 | Viewed by 3608

Abstract

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has [...] Read more.

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has been associated with many disease states. To investigate miRNA expression in host cells during H. capsulatum infection, we studied the changes in the miRNA profiles of differentiated human macrophages infected with yeasts from two fungal strains with different virulence, EH-315 (high virulence) and 60I (low virulence) grown in planktonic cultures, and EH-315 grown in biofilm form. MiRNA profiles were evaluated by means of reverse transcription-quantitative polymerase chain reaction using a commercial human miRNome panel. The target genes of the differentially expressed miRNAs and their corresponding signaling pathways were predicted using bioinformatics analyses. Here, we confirmed biofilm structures were present in the EH-315 culture whose conditions facilitated producing insoluble exopolysaccharide and intracellular polysaccharides. In infected macrophages, bioinformatics analyses revealed especially increased (hsa-miR-99b-3p) or decreased (hsa-miR-342-3p) miRNAs expression levels in response to infection with biofilms or both growth forms of H. capsulatum yeasts, respectively. The results of miRNAs suggested that infection by H. capsulatum can affect important biological pathways of the host cell, targeting two genes: one encoding a protein that is important in the cortical cytoskeleton; the other, a protein involved in the formation of stress granules. Expressed miRNAs in the host’s response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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11 pages, 985 KiB

Open AccessArticle

Paracoccidioides brasiliensis Isolated from Nine-Banded Armadillos (Dasypus novemcinctus) Reveal Population Structure and Admixture in the Amazon Basin

by Eduardo Bagagli, Daniel Ricardo Matute, Hans Garcia Garces, Bernardo Guerra Tenório, Adalberto Garcia Garces, Lucas Gomes de Brito Alves, Danielle Hamae Yamauchi, Marluce Francisca Hrycyk, Bridget Marie Barker and Marcus de Melo Teixeira

J. Fungi 2021, 7(1), 54; https://doi.org/10.3390/jof7010054 - 15 Jan 2021

Cited by 5 | Viewed by 2986

Abstract

Paracoccidioidomycosis is an endemic fungal disease to Latin America caused by at least five species-level genotypes of Paracoccidioides, named P. lutzii, P. brasiliensis (S1a and S1b populations), P. americana, P. restrepiensis, and P. venezuelensis. In this manuscript, we [...] Read more.

Paracoccidioidomycosis is an endemic fungal disease to Latin America caused by at least five species-level genotypes of Paracoccidioides, named P. lutzii, P. brasiliensis (S1a and S1b populations), P. americana, P. restrepiensis, and P. venezuelensis. In this manuscript, we report on Paracoccidioides sp. sampling efforts in armadillos from two different areas in Brazil. We sequenced the genomes of seven Paracoccidioides isolates and used phylogenomics and populations genetics for genotyping. We found that P. brasiliensis and P. lutzii are both present in the Amazon region. Additionally, we identified two Paracoccidioides isolates that seem to be the result of admixture between divergent populations within P. brasiliensis sensu stricto. Both of these isolates were recovered from armadillos in a P. lutzii endemic area in Midwestern Brazil. Additionally, two isolates from human patients also show evidence of resulting from admixture. Our results suggest that the populations of P. brasiliensis sensu stricto exchange genes in nature. More generally, they suggest that population structure and admixture within species is an important source of variation for pathogenic fungi. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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16 pages, 1291 KiB

Open AccessArticle

In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein

by Liliana Scorzoni, Ana Carolina Alves de Paula e Silva, Haroldo Cesar de Oliveira, Claudia Tavares dos Santos, Junya de Lacorte Singulani, Patricia Akemi Assato, Caroline Maria Marcos, Lariane Teodoro Oliveira, Nathália Ferreira Fregonezi, Diego Conrado Pereira Rossi, Leandro Buffoni Roque da Silva, Carlos Pelleschi Taborda, Ana Marisa Fusco-Almeida and Maria José Soares Mendes-Giannini

J. Fungi 2021, 7(1), 52; https://doi.org/10.3390/jof7010052 - 13 Jan 2021

Cited by 5 | Viewed by 2884

Abstract

Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in [...] Read more.

Background: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. Methods: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. Results: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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21 pages, 2863 KiB

Open AccessArticle

Prediction of Conserved Peptides of Paracoccidioides for Interferon-γ Release Assay: The First Step in the Development of a Lab-Based Approach for Immunological Assessment during Antifungal Therapy

by Sarah Brena Aparecida Rosa, Bárbara Guimarães Csordas, Sandra Maria do Valle Leone de Oliveira, Amanda Ribeiro dos Santos, Anamaria Mello Miranda Paniago and James Venturini

J. Fungi 2020, 6(4), 379; https://doi.org/10.3390/jof6040379 - 19 Dec 2020

Cited by 4 | Viewed by 2992

Abstract

Impaired antigen-specific cell-mediated immunity (CMI) is a primary immunological disturbance observed in individuals that develop paracoccidioidomycosis (PCM) after exposure to Paracoccidioides spp. Restoration of Paracoccidioides-specific CMI is crucial to stop the antifungal treatment and avoid relapses. A convenient and specific laboratory tool [...] Read more.

Impaired antigen-specific cell-mediated immunity (CMI) is a primary immunological disturbance observed in individuals that develop paracoccidioidomycosis (PCM) after exposure to Paracoccidioides spp. Restoration of Paracoccidioides-specific CMI is crucial to stop the antifungal treatment and avoid relapses. A convenient and specific laboratory tool to assess antigen specific CMI is required for the appropriate clinical treatment of fungal infections, in order to decrease the time of antifungal therapy. We used an interferon-γ release assay strategy, used in the diagnosis of latent tuberculosis infection, to address our aims in this study. Information on proteins secreted by two well-studied representative strains—Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb-01)—were explored using PubMed or MEDLINE. From 26 publications, 252 proteins were identified, of which 203 were similar according to the Basic Local Alignment Search Tool. This enabled a selection of conserved peptides using the MEGA software. The SignalP-5.0, TMHMM, IEDB, NetMHC II, and IFNepitope algorithms were used to identify appropriate epitopes. In our study, we predicted antigenic epitopes of Paracoccidioides that could bind to MHC class II and induce IFN-γ secretion. These T cell epitopes can be used in the development of a laboratory tool to monitor the CMI of patients with PCM. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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36 pages, 5893 KiB

Open AccessArticle

Immunoproteomic Analysis Reveals Novel Candidate Antigens for the Diagnosis of Paracoccidioidomycosis Due to Paracoccidioides lutzii

by Anderson Messias Rodrigues, Paula Helena Kubitschek-Barreira, Breno Gonçalves Pinheiro, André Teixeira-Ferreira, Rosane Christine Hahn and Zoilo Pires de Camargo

J. Fungi 2020, 6(4), 357; https://doi.org/10.3390/jof6040357 - 11 Dec 2020

Cited by 21 | Viewed by 4994

Abstract

Paracoccidioidomycosis (PCM) is a life-threatening systemic infection caused by the fungal pathogen Paracoccidioides brasiliensis and related species. Whole-genome sequencing and stage-specific proteomic analysis of Paracoccidioides offer the opportunity to profile humoral immune responses against P. lutzii and P. brasiliensis s. str. infection using [...] Read more.

Paracoccidioidomycosis (PCM) is a life-threatening systemic infection caused by the fungal pathogen Paracoccidioides brasiliensis and related species. Whole-genome sequencing and stage-specific proteomic analysis of Paracoccidioides offer the opportunity to profile humoral immune responses against P. lutzii and P. brasiliensis s. str. infection using innovative screening approaches. Here, an immunoproteomic approach was used to identify PCM-associated antigens that elicit immune responses by combining 2-D electrophoresis of P. lutzii and P. brasiliensis proteomes, immunological detection using a gold-standard serum, and mass spectrometry analysis. A total of 16 and 25 highly immunoreactive proteins were identified in P. lutzii and P. brasiliensis, respectively, and 29 were shown to be the novel antigens for Paracoccidioides species, including seven uncharacterized proteins. Among the panel of proteins identified, most are involved in metabolic pathways, carbon metabolism, and biosynthesis of secondary metabolites in both immunoproteomes. Remarkably, six isoforms of the surface-associated enolase in the range of 54 kDa were identified as the major antigens in human PCM due to P. lutzii. These novel immunoproteomes of Paracoccidioides will be employed to develop a sensitive and affordable point-of-care diagnostic assay and an effective vaccine to identify infected hosts and prevent infection and development of human PCM. These findings provide a unique opportunity for the refinement of diagnostic tools of this important neglected systemic mycosis, which is usually associated with poverty. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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16 pages, 2323 KiB

Open AccessArticle

Therapeutic Vaccination with Cationic Liposomes Formulated with Dioctadecyldimethylammonium and Trehalose Dibehenate (CAF01) and Peptide P10 Is Protective in Mice Infected with Paracoccidioides brasiliensis

by Marcelo Valdemir de Araújo, Samuel Rodrigues Dos Santos Júnior, Joshua D. Nosanchuk and Carlos Pelleschi Taborda

J. Fungi 2020, 6(4), 347; https://doi.org/10.3390/jof6040347 - 8 Dec 2020

Cited by 5 | Viewed by 2388

Abstract

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated [...] Read more.

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4⁺ Th₁ lymphocytes that was associated with the increased production of IFN-γ in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01–cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th₁/Th₁₇ cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 µg/mL and 20 µg of P10 induced an effective cellular immune response with increased levels of Th₁₇ cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 µg/mL with 20 µg of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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18 pages, 4516 KiB

Open AccessArticle

Beyond Melanin: Proteomics Reveals Virulence-Related Proteins in Paracoccidioides brasiliensis and Paracoccidioides lutzii Yeast Cells Grown in the Presence of L-Dihydroxyphenylalanine

by Rodrigo Almeida-Paes, Marcos Abreu Almeida, Lilian Cristiane Baeza, Leticia Andrade Mendes Marmello, Monique Ramos de Oliveira Trugilho, Joshua Daniel Nosanchuk, Celia Maria de Almeida Soares, Richard Hemmi Valente and Rosely Maria Zancopé-Oliveira

J. Fungi 2020, 6(4), 328; https://doi.org/10.3390/jof6040328 - 1 Dec 2020

Cited by 5 | Viewed by 2254

Abstract

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen’s survival in the host. To better understand the metabolic changes that [...] Read more.

Species of the genus Paracoccidioides cause a systemic infection in human patients. Yeast cells of Paracoccidioides spp. produce melanin in the presence of L-dihydroxyphenylalanine and during infection, which may impact the pathogen’s survival in the host. To better understand the metabolic changes that occur in melanized Paracoccidioides spp. cells, a proteomic approach was performed to compare melanized and non-melanized Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells. Melanization was induced using L-dihydroxyphenylalanine as a precursor, and quantitative proteomics were performed using reversed-phase nano-chromatography coupled to high-resolution mass spectrometry. When comparing melanized versus non-melanized cells, 1006 and 582 differentially abundant/detected proteins were identified for P. brasiliensis and P. lutzii, respectively. Functional enrichment and comparative analysis revealed 30 important KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways in melanized P. brasiliensis and 18 in P. lutzii, while differentially abundant proteins from non-melanized cells from these species were involved in 21 and 25 enriched pathways, respectively. Melanized cells presented an abundance of additional virulence-associated proteins, such as phospholipase, proteases, superoxide dis-mutases, heat-shock proteins, adhesins, and proteins related to vesicular transport. The results suggest that L-dihydroxyphenylalanine increases the virulence of Paracoccidioides spp. through complex mechanisms involving not only melanin but other virulence factors as well. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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22 pages, 5746 KiB

Open AccessArticle

Transcriptional Remodeling Patterns in Murine Dendritic Cells Infected with Paracoccidioides brasiliensis: More Is Not Necessarily Better

by Calliandra M. de-Souza-Silva, Fabián Andrés Hurtado, Aldo Henrique Tavares, Getúlio P. de Oliveira, Jr., Taina Raiol, Christiane Nishibe, Daniel Paiva Agustinho, Nalvo Franco Almeida, Maria Emília Machado Telles Walter, André Moraes Nicola, Anamélia Lorenzetti Bocca, Patrícia Albuquerque and Ildinete Silva-Pereira

J. Fungi 2020, 6(4), 311; https://doi.org/10.3390/jof6040311 - 24 Nov 2020

Cited by 2 | Viewed by 2491

Abstract

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) [...] Read more.

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway’s repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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23 pages, 3146 KiB

Open AccessArticle

Interaction of Isocitrate Lyase with Proteins Involved in the Energetic Metabolism in Paracoccidioides lutzii

by Kleber Santiago Freitas e Silva, Raisa Melo Lima, Patrícia de Sousa Lima, Lilian Cristiane Baeza, Roosevelt Alves da Silva, Célia Maria de Almeida Soares and Maristela Pereira

J. Fungi 2020, 6(4), 309; https://doi.org/10.3390/jof6040309 - 23 Nov 2020

Cited by 2 | Viewed by 2411

Abstract

Background: Systemic mycosis is a cause of death of immunocompromised subjects. The treatment directed to evade fungal pathogens shows severe limitations, such as time of drug exposure and side effects. The paracoccidioidomycosis (PCM) treatment depends on the severity of the infection and may [...] Read more.

Background: Systemic mycosis is a cause of death of immunocompromised subjects. The treatment directed to evade fungal pathogens shows severe limitations, such as time of drug exposure and side effects. The paracoccidioidomycosis (PCM) treatment depends on the severity of the infection and may last from months to years. Methods: To analyze the main interactions of Paracoccidioides lutzii isocitrate lyase (ICL) regarding the energetic metabolism through affinity chromatography, we performed blue native PAGE and co-immunoprecipitation to identify ICL interactions. We also performed in silico analysis by homology, docking, hot-spot prediction and contact preference analysis to identify the conformation of ICL complexes. Results: ICL interacted with 18 proteins in mycelium, 19 in mycelium-to-yeast transition, and 70 in yeast cells. Thirty complexes were predicted through docking and contact preference analysis. ICL has seven main regions of interaction with protein partners. Conclusions: ICL seems to interfere with energetic metabolism of P. lutzii, regulating aerobic and anaerobic metabolism as it interacts with proteins from glycolysis, gluconeogenesis, TCA and methylcitrate cycles, mainly through seven hot-spot residues. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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8 pages, 539 KiB

Open AccessCommunication

Neuroparacoccidioidomycosis: A 13-Year Cohort Study, Rio de Janeiro, Brazil

by Priscila Marques de Macedo, Eduardo Mastrangelo Marinho Falcão, Dayvison Francis Saraiva Freitas, Andréa d’Avila Freitas, Ziadir Francisco Coutinho, Mauro de Medeiros Muniz, Rosely Maria Zancopé-Oliveira, Rodrigo Almeida-Paes, Marcus Tulius Teixeira da Silva and Antonio Carlos Francesconi do Valle

J. Fungi 2020, 6(4), 303; https://doi.org/10.3390/jof6040303 - 20 Nov 2020

Cited by 4 | Viewed by 1974

Abstract

Neuroparacoccidioidomycosis (NPCM) is a rare and severe clinical presentation of paracoccidioidomycosis (PCM). We performed a retrospective cohort study at the Evandro Chagas National Institute of Infectious Diseases (INI/Fiocruz), a reference center for PCM in the state of Rio de Janeiro, Brazil. All cases [...] Read more.

Neuroparacoccidioidomycosis (NPCM) is a rare and severe clinical presentation of paracoccidioidomycosis (PCM). We performed a retrospective cohort study at the Evandro Chagas National Institute of Infectious Diseases (INI/Fiocruz), a reference center for PCM in the state of Rio de Janeiro, Brazil. All cases of PCM admitted to the INI/Fiocruz from January 2007 to December 2019 were reviewed. Eight (3.9%) among 207 patients met the diagnostic criteria for NPCM. The mean age was 44.6 years and the male:female ratio was 7:1. All cases presented multifocal disease, 5 (62.5%) the chronic form and 3 (37.5%) the acute/subacute form. All patients presented the pseudotumoral pattern and 6 (75.0%) had multiple lesions in the cerebral hemispheres. Seizures and motor symptoms were the most frequent clinical manifestations (50.0%, each). The treatment of choice was sulfamethoxazole/trimethoprim (SMZ-TMP) and fluconazole, in association (87.5%). Most patients responded well to the treatment. Sequela and death occurred in one (12.5%) patient, each. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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11 pages, 2337 KiB

Open AccessArticle

Melanin as a Virulence Factor in Different Species of Genus Paracoccidioides

by Elúzia C. P. Emidio, Martha E. Urán J., Leandro B. R. Silva, Lucas S. Dias, Mariana Doprado, Joshua D. Nosanchuk and Carlos Pelleschi Taborda

J. Fungi 2020, 6(4), 291; https://doi.org/10.3390/jof6040291 - 17 Nov 2020

Cited by 5 | Viewed by 2711

Abstract

Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis caused by the thermo-dimorphic fungi of the genus Paracoccidioides. Melanin production by fungi can affect their pathogenesis and virulence. This study evaluates the production of melanin by different isolates of genus Paracoccidioides and examines how [...] Read more.

Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis caused by the thermo-dimorphic fungi of the genus Paracoccidioides. Melanin production by fungi can affect their pathogenesis and virulence. This study evaluates the production of melanin by different isolates of genus Paracoccidioides and examines how the presence of this polymer affects yeast cell phagocytosis, as well as laccase enzyme production. The results obtained showed that the isolates of genus Paracoccidioides: P. lutzii (Pb01, Pb66, ED01, Pb1578, and Pb8334), P. restrepiensis (PS3-Pb60855), P. brasiliensis (S1-Pb18), and P. americana (PS2-Pbcão) produce melanin in the presence of L-3,4-dihydroxyphenylalanine (L-DOPA). Phagocytosis assays were carried out with peritoneal macrophages from C57Bl/6 mice that were challenged with Pb18, Pb60855, and Pb01. We observed that melanin interferes with phagocytosis in the presence or absence of complement or heat-inactivated serum. This article confirms that different species of the genus Paracoccidioides produce melanin in different magnitudes and that the polymer functions as a virulence factor. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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12 pages, 15992 KiB

Open AccessArticle

Phylogenetic Species of Paracoccidioides spp. Isolated from Clinical and Environmental Samples in a Hyperendemic Area of Paracoccidioidomycosis in Southeastern Brazil

by Tiago A. Cocio, Erika Nascimento, Marcia R. von Zeska Kress, Eduardo Bagagli and Roberto Martinez

J. Fungi 2020, 6(3), 132; https://doi.org/10.3390/jof6030132 - 11 Aug 2020

Cited by 11 | Viewed by 2574

Abstract

Paracoccidioides brasiliensis complex and P. lutzii are the etiological agents of paracoccidioidomycosis. The geographic distribution of these species in South America is still poorly comprehended. Fifty samples of Paracoccidioides spp. were genotyped, with 46 clinical isolates predominantly isolated in the geographic area of [...] Read more.

Paracoccidioides brasiliensis complex and P. lutzii are the etiological agents of paracoccidioidomycosis. The geographic distribution of these species in South America is still poorly comprehended. Fifty samples of Paracoccidioides spp. were genotyped, with 46 clinical isolates predominantly isolated in the geographic area of Ribeirão Preto, SP, and four environmental isolates collected in Ibiá, MG, southeastern Brazil. These isolates were evaluated by PCR-RFLP (Restriction Fragment Length Polymorphism) of the tub1 gene and the sequencing of the gp43 exon 2 loci. The species P. lutzii was confirmed by sequencing the internal transcribed spacer (ITS) region of the ribosomal DNA. P. brasiliensis sensu stricto S1b (n = 42) and S1a (n = 5), P. americana (n = 1), P. restrepiensis (n = 1), and P. lutzii (n = 1) were identified among the clinical isolates. All the environmental isolates were characterized as P. brasiliensis sensu stricto S1b. The patient infection by P. lutzii, P. americana (PS2), and one isolate of P. brasiliensis sensu stricto S1b most likely occurred in a geographic area far from the fungal isolation site. No association was found between the infecting genotype and the disease form. These results expand the knowledge of the Paracoccidioides species distribution and emphasize that human migration must also be considered to pinpoint the genotypes in the endemic area. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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Review

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25 pages, 402 KiB

Open AccessReview

Paracoccidioidomycosis Protective Immunity

by Eva Burger

J. Fungi 2021, 7(2), 137; https://doi.org/10.3390/jof7020137 - 13 Feb 2021

Cited by 15 | Viewed by 3218

Abstract

Protective immunity against Paracoccidioides consists of a stepwise activation of numerous effector mechanisms that comprise many cellular and soluble components. At the initial phase of non-specific innate immunity, resistance against Paracoccidioides comes from phagocytic polymorphonuclear neutrophils, natural killer (NK) cells and monocytes, supplemented [...] Read more.

Protective immunity against Paracoccidioides consists of a stepwise activation of numerous effector mechanisms that comprise many cellular and soluble components. At the initial phase of non-specific innate immunity, resistance against Paracoccidioides comes from phagocytic polymorphonuclear neutrophils, natural killer (NK) cells and monocytes, supplemented by soluble factors such as cytokines and complement system components. Invariant receptors (Toll-like receptors (TLRs), Dectins) which are present in cells of the immune system, detect patterns present in Paracoccidioides (but not in the host) informing the hosts cells that there is an infection in progress, and that the acquired immunity must be activated. The role of components involved in the innate immunity of paracoccidioidomycosis is herein presented. Humoral immunity, represented by specific antibodies which control the fungi in the blood and body fluids, and its role in paracoccidioidomycosis (which was previously considered controversial) is also discussed. The protective mechanisms (involving various components) of cellular immunity are also discussed, covering topics such as: lysis by activated macrophages and cytotoxic T lymphocytes, the participation of lytic products, and the role of cytokines secreted by T helper lymphocytes in increasing the efficiency of Paracoccidioides, lysis. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

26 pages, 2572 KiB

Open AccessReview

One Century of Study: What We Learned about Paracoccidioides and How This Pathogen Contributed to Advances in Antifungal Therapy

by Erika Seki Kioshima, Patrícia de Souza Bonfim de Mendonça, Marcus de Melo Teixeira, Isis Regina Grenier Capoci, André Amaral, Franciele Abigail Vilugron Rodrigues-Vendramini, Bruna Lauton Simões, Ana Karina Rodrigues Abadio, Larissa Fernandes Matos and Maria Sueli Soares Felipe

J. Fungi 2021, 7(2), 106; https://doi.org/10.3390/jof7020106 - 2 Feb 2021

Cited by 3 | Viewed by 3095

Abstract

Paracoccidioidomycosis (PCM) is a notable fungal infection restricted to Latin America. Since the first description of the disease by Lutz up to the present day, Brazilian researchers have contributed to the understanding of the life cycle of this pathogen and provided the possibility [...] Read more.

Paracoccidioidomycosis (PCM) is a notable fungal infection restricted to Latin America. Since the first description of the disease by Lutz up to the present day, Brazilian researchers have contributed to the understanding of the life cycle of this pathogen and provided the possibility of new targets for antifungal therapy based on the structural and functional genomics of Paracoccidioides. In this context, in silico approaches have selected molecules that act on specific targets, such as the thioredoxin system, with promising antifungal activity against Paracoccidioides. Some of these are already in advanced development stages. In addition, the application of nanostructured systems has addressed issues related to the high toxicity of conventional PCM therapy. Thus, the contribution of molecular biology and biotechnology to the advances achieved is unquestionable. However, it is still necessary to transcend the boundaries of synthetic chemistry, pharmaco-technics, and pharmacodynamics, aiming to turn promising molecules into newly available drugs for the treatment of fungal diseases. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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21 pages, 1491 KiB

Open AccessReview

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

by Lívia do Carmo Silva, Amanda Alves de Oliveira, Dienny Rodrigues de Souza, Katheryne Lohany Barros Barbosa, Kleber Santiago Freitas e Silva, Marcos Antonio Batista Carvalho Júnior, Olívia Basso Rocha, Raisa Melo Lima, Thaynara Gonzaga Santos, Célia Maria de Almeida Soares and Maristela Pereira

J. Fungi 2020, 6(4), 300; https://doi.org/10.3390/jof6040300 - 19 Nov 2020

Cited by 19 | Viewed by 3520

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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24 pages, 13773 KiB

Open AccessReview

Molecular Tools for Detection and Identification of Paracoccidioides Species: Current Status and Future Perspectives

by Breno Gonçalves Pinheiro, Rosane Christine Hahn, Zoilo Pires de Camargo and Anderson Messias Rodrigues

J. Fungi 2020, 6(4), 293; https://doi.org/10.3390/jof6040293 - 18 Nov 2020

Cited by 28 | Viewed by 5624

Abstract

Paracoccidioidomycosis (PCM) is a mycotic disease caused by the Paracoccidioides species, a group of thermally dimorphic fungi that grow in mycelial form at 25 °C and as budding yeasts when cultured at 37 °C or when parasitizing the host tissues. PCM occurs in [...] Read more.

Paracoccidioidomycosis (PCM) is a mycotic disease caused by the Paracoccidioides species, a group of thermally dimorphic fungi that grow in mycelial form at 25 °C and as budding yeasts when cultured at 37 °C or when parasitizing the host tissues. PCM occurs in a large area of Latin America, and the most critical regions of endemicity are in Brazil, Colombia, and Venezuela. The clinical diagnosis of PCM needs to be confirmed through laboratory tests. Although classical laboratory techniques provide valuable information due to the presence of pathognomonic forms of Paracoccidioides spp., nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratory practice. Recently, taxonomic changes driven by whole-genomic sequencing of Paracoccidioides have highlighted the need to recognize species boundaries, which could better ascertain Paracoccidioides taxonomy. In this scenario, classical laboratory techniques do not have significant discriminatory power over cryptic agents. On the other hand, several PCR-based methods can detect polymorphisms in Paracoccidioides DNA and thus support species identification. This review is focused on the recent achievements in molecular diagnostics of paracoccidioidomycosis, including the main advantages and pitfalls related to each technique. We discuss these breakthroughs in light of taxonomic changes in the Paracoccidioides genus. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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12 pages, 1113 KiB

Open AccessReview

The “Little Iron Waltz”: The Ternary Response of Paracoccidioides spp. to Iron Deprivation

by Aparecido Ferreira de Souza, Marcella Silva de Paula, Raisa Melo Lima, Marielle Garcia Silva, Juliana Santana de Curcio, Maristela Pereira and Célia Maria de Almeida Soares

J. Fungi 2020, 6(4), 221; https://doi.org/10.3390/jof6040221 - 12 Oct 2020

Cited by 1 | Viewed by 2327

Abstract

Paracoccidioides is a genus of thermodimorphic fungi that causes paracoccidioidomycosis. When in the host, the fungus undergoes several challenges, including iron deprivation imposed by nutritional immunity. In response to the iron deprivation triggered by the host, the fungus responds in a ternary manner [...] Read more.

Paracoccidioides is a genus of thermodimorphic fungi that causes paracoccidioidomycosis. When in the host, the fungus undergoes several challenges, including iron deprivation imposed by nutritional immunity. In response to the iron deprivation triggered by the host, the fungus responds in a ternary manner using mechanisms of high affinity and specificity for the uptake of Fe, namely non-classical reductive iron uptake pathway, uptake of host iron proteins, and biosynthesis and uptake of siderophores. This triple response resembles the rhythmic structure of a waltz, which features three beats per compass. Using this connotation, we have constructed this review summarizing relevant findings in this area of study and pointing out new discoveries and perspectives that may contribute to the expansion of this “little iron waltz”. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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14 pages, 6552 KiB

Open AccessReview

The Therapy of Pulmonary Fibrosis in Paracoccidioidomycosis: What Are the New Experimental Approaches?

by Ángel González

J. Fungi 2020, 6(4), 217; https://doi.org/10.3390/jof6040217 - 11 Oct 2020

Cited by 10 | Viewed by 3156

Abstract

Pulmonary fibrosis (PF) is considered the most important sequela developed in patients suffering from the chronic form of paracoccidioidomycosis (PCM), which leads to the loss of respiratory function in 50% of cases; this residual pulmonary abnormality is present even after antifungal treatment. To [...] Read more.

Pulmonary fibrosis (PF) is considered the most important sequela developed in patients suffering from the chronic form of paracoccidioidomycosis (PCM), which leads to the loss of respiratory function in 50% of cases; this residual pulmonary abnormality is present even after antifungal treatment. To date, there is no effective treatment for PF. However, the use of antifungal drugs in combination with other antibiotics or immunomodulatory compounds, as well as biological therapies that include a monoclonal antibody specific to neutrophils, or prophylactic vaccination employing a recombinant antigen of Paracoccidioides brasiliensis that successfully attenuated PF, has been reported. Additionally, mesenchymal stem cell transplantation in combination with antifungal therapy slightly reduced the inflammatory response and profibrotic molecules induced by P. brasiliensis infection. In this review, I report experimental findings from several studies aiming to identify promising therapeutic strategies for treating PF developed in PCM. Full article

(This article belongs to the Special Issue Paracoccidioides and Paracoccidioidomycosis)

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