Fungal Extracellular Vesicles Are Involved in Intraspecies Intracellular Communication

Here, we report a study about extracellular vesicles (EVs) as communication mediators in fungi. Our results demonstrated the role of EVs from Candida albicans , Aspergillus fumigatus , and Paracoccidioides brasiliensis regulating the expression of target genes and phenotype features.

Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development

Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181–195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

Brain paracoccidioidomycosis in an immunosuppressed patient with systemic lupus erythematosus

Background: Brain paracoccidioidomycosis (PCM) or neuroparacoccidioidomycosis (NPCM) is a fungal infection of the central nervous system (CNS) caused by Paracoccidioides brasiliensis, a dimorphic fungus. The CNS involvement is through bloodstream dissemination. The association between NPCM and systemic lupus erythematous (SLE) is rare. However, SLE patients are under risk of opportunistic infections given their immunosuppression status. Case Description: The aim of this case report is to present a 37-year-old female with diagnosis of SLE who presented with progressive and persistent headache in the past 4 months accompanied by the right arm weakness with general and neurologic examination unremarkable. The computerized tomography of the head showed left extra-axial parietooccipital focal hypoattenuation with adjacent bone erosion. The brain magnetic resonance imaging reported left parietooccipital subdural collection associated with focal leptomeningeal thickening with restriction to diffusion and peripheral contrast enhancement. The patient underwent a left craniotomy and dura mater biopsy showed noncaseous granulomatosis with multinucleated giant cells with rounded birefringent structures positive for silver stain, consistent with PCM. Management with itraconazole 200 mg daily was started with a total of 12 months of treatment, with patient presenting resolution of headache and right arm weakness. Conclusion: The diagnosis of NPCM is challenging and a high degree of suspicious should be considered in patients with persistent headache and immunosuppression.

The Role of Dimorphism Regulating Histidine Kinase (Drk1) in the Pathogenic Fungus Paracoccidioides brasiliensis Cell Wall

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a β-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher β-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify β-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals.

Paracoccidioidomicose em cão – relato de caso / Paracoccidioidomycosis in dog - case report

A paracoccidioidomicose é uma infecção fúngica sistêmica, endêmica, causada pelo fungo dimórfico paracoccidioides brasiliensis. Entre os principais sintomas apresentados estão inapetência, fraqueza, emagrecimento, palidez, febre, dispnéia, tosse e lesões na pele ou mucosas. O diagnóstico na medicina veterinária de pequenos animais é um desafio devido aos poucos relatos em literatura da doença nestes pacientes e consequentemente o pouco conhecimento da patologia pelos clínicos. O presente trabalho relata um caso de paracoccidioidomicose em cão atendido em Curitiba-PR, e diagnosticado através de exames de sangue e cultura fúngica sanguínea. O tratamento instaurado foi eficaz. O relato mostra a importância da consideração da paracoccidioidomicose nos diagnósticos diferenciais em cães.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro

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.

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

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.

Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development

Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that are capable of forming amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

PbGP43 Genotyping Using Paraffin-embedded Biopsies of Human Paracoccidioidomycosis Reveals a Genetically Distinct Lineage in the Paracoccidioides Brasiliensis Complex

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher.This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection.Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins.Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein.This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.