Sbeghen, Mônica Raquel; Zanata, Thais Bastos; Macagnan, Rafaela; de Abreu, Kaue Cachuba; da Cunha, Willian Luiz; Watanabe, Maria Angelica Ehara; de Camargo, Zoilo Pires; Ono, Mario Augusto
Paracoccidioidomycosis (PCM) is a systemic mycosis prevalent in Brazil and other Latin American countries. The etiological agents of PCM are the thermo-dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. Taking into account that the natural habitat of Paracoccidioides spp. is still undefined, domestic and wild animals could be useful as indicators of Paracoccidioides spp. presence in endemic areas. The objective of this study was to evaluate the infection of small wild mammals by P. brasiliensis in an endemic area for human PCM. Samples from 38 wild mammals from different species such as Akodon sp., Thaptomys nigrita, Euryoryzomys russatus, Oligoryzomys nigripes, Monodelphis sp., Sooretamys angouya, Abrawayaomys angouya, Abrawayaomys ruschii and Akodontinae sp. were evaluated by ELISA, immunodiffusion, histopathology, nested PCR and culture. The overall positivity to gp43 observed in the ELISA was 23.7%. Samples from heart and liver of one O. nigripes were PCR positive, and the animal was also seropositive to gp43 in ELISA. This study showed that wild animals living in endemic areas for PCM are infected with P. brasiliensis and can be valuable epidemiological markers of the fungus presence in the environment. This is the first evidence of PCM infection in Akodon sp., E. russatus, T. nigrita and O. nigripes.
de Oliveira, Haroldo C.; Michaloski, Jussara S.; da Silva, Julhiany F.; Scorzoni, Liliana; de Paula e Silva, Ana C. A.; Marcos, Caroline M.; Assato, Patrícia A.; Yamazaki, Daniella S.; Fusco-Almeida, Ana M.; Giordano, Ricardo J.; Mendes-Giannini, Maria J. S.
Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy. PMID:28066254
de Oliveira, Haroldo C; Michaloski, Jussara S; da Silva, Julhiany F; Scorzoni, Liliana; de Paula E Silva, Ana C A; Marcos, Caroline M; Assato, Patrícia A; Yamazaki, Daniella S; Fusco-Almeida, Ana M; Giordano, Ricardo J; Mendes-Giannini, Maria J S
Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy.
Albano, Ana Paula Neuschrank; Klafke, Gabriel Baracy; Brandolt, Tchana Martinez; Da Hora, Vanusa Pousada; Nogueira, Carlos Eduardo Wayne; Xavier, Melissa Orzechowski; Meireles, Mário Carlos Araújo
Paracoccidioides brasiliensis is the etiological agent of the major systemic mycosis in Brazil, called paracoccidioidomycosis. Although the Rio Grande do Sul is considered an endemic area of the disease, there are few studies on the ecology of P. brasiliensis in the state. Therefore, this study aimed to evaluate the infection of P. brasiliensis in horses from the mesoregion of Southwest Riograndense, using these animals as sentinels. Serological techniques, such as double immunodiffusion in agar gel (AGID) and indirect ELISA, were performed to detect the anti-gp43 P. brasiliensis antibody in horses from five different farms in the region of Bagé, RS, Brazil. Serology was performed in 200 Pure Blood English horses up to two years of age that were born and raised exclusively at the farms. Of these horses, 12% had anti-gp43 antibodies according to the ELISA results, with rates ranging from 0 to 30% according to the farm of origin (p < 0.001). Based on the immunodiffusion results, all equine serum samples were negative. These results indicate the presence of the fungus P. brasiliensis in the middle region of the southwestern state of Rio Grande do Sul, Brazil. PMID:26273267
Urán, Martha E; Nosanchuk, Joshua D; Restrepo, Angela; Hamilton, Andrew J; Gómez, Beatriz L; Cano, Luz E
Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice.
Restrepo, S; Tobon, A; Trujillo, J; Restrepo, A
Pulmonary fibrosis is a feared sequelae of paracoccidioidomycosis. We sought to determine if mice exposed to Paracoccidioides brasiliensis conidia would develop pulmonary fibrosis. BALB/c mice were infected intranasally with P. brasiliensis conidia and sacrificed at regular intervals. One lung was sectioned for histopathology and sections were stained with haematoxylin and eosin, trichromic and argentic stains; the other lung was homogenized and cultured to determine the viability of the fungus. One week post-challenge, only small peribronchial foci were apparent. After 4 weeks, reticular fibres appeared disorganized and disrupted. Six to 8 weeks later peribronchial infiltrates were larger and appeared surrounded by reticular fibres; thick collagen I fibres were noticed in the infiltrated areas at this time. On weeks 10-12, infiltrates were confluent and reticular fibres were concentrated around the inflammatory foci; collagenization was apparent. Observations up to 16 weeks revealed diffuse involvement of the lung parenchyma with extensive collagenization. Lung cultures were always positive. We suggest that inhalation of P. brasiliensis conidia induces adverse lung responses leading to changes in the proportion of collagen fibres I and III.
Fernandes, Fabrício Freitas; de Oliveira, Leandro Licursi; Landgraf, Taise Natali; Peron, Gabriela; Costa, Marcelo Vieira; Coelho-Castelo, Arlete A. M.; Bonato, Vânia L. D.; Roque-Barreira, Maria-Cristina; Panunto-Castelo, Ademilson
The genus Paracoccidioides comprises species of dimorphic fungi that cause paracoccidioidomycosis (PCM), a systemic disease prevalent in Latin America. Here, we investigated whether administration of native 60-kDa heat shock protein of P. brasiliensis (nPbHsp60) or its recombinant counterpart (rPbHsp60) affected the course of experimental PCM. Mice were subcutaneously injected with nPbHsp60 or rPbHsp60 emulsified in complete’s Freund Adjuvant (CFA) at three weeks after intravenous injection of P. brasiliensis yeasts. Infected control mice were injected with CFA or isotonic saline solution alone. Thirty days after the nPbHsp60 or rPbHsp60 administration, mice showed remarkably increased fungal load, tissue inflammation, and granulomas in the lungs, liver, and spleen compared with control mice. Further, rPbHsp60 treatment (i) decreased the known protective effect of CFA against PCM and (ii) increased the concentrations of IL-17, TNF-α, IL-12, IFN-γ, IL-4, IL-10, and TGF-β in the lungs. Together, our results indicated that PbHsp60 induced a harmful immune response, exacerbated inflammation, and promoted fungal dissemination. Therefore, we propose that PbHsp60 contributes to the fungal pathogenesis. PMID:27598463
Bueno, Renata A; Thomaz, Luciana; Muñoz, Julian E; da Silva, Cássia J; Nosanchuk, Joshua D; Pinto, Márcia R; Travassos, Luiz R; Taborda, Carlos P
Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.
Martins, Jaciara F. S.; Castilho, Maiara L.; Cardoso, Maria A. G.; Carreiro, Andrea P.; Martin, Airton A.; Raniero, Leandro
Paracoccidioides brasiliensis (P. brasiliensis) is a thermal dimorphic fungus and causal agent of paracoccidioidomycosis. Epidemiological data shows that it is mainly concentrated in Central and South America countries, with most registered cases in Colombia, Brazil, and Venezuela. The histopathological similarity with others fungal infection makes the diagnosis of P. brasiliensis more complicated. Therefore, the aim of this work was to find a positive and negative test for P. brasiliensis using gold nanoprobes as a new tool for P. brasiliensis detection. Gold nanoparticles were synthesized by reduction of gold chloride with sodium citrate. The results of this procedure is a wine-red solution with a maximum absorption in the range of ~520-530nm. A specific P. brasiliensis sequence of oligonucleotide was bonded to the nanoparticles, which maintained the wine-red color. The color changes from red to blue for negative diagnostic and is unchanged for a positive test. The H-bond interaction of DNA with the complementary DNA keeps strands together and forms double helical structure, maintaining the colloid stability. However, for non-complimentary DNA sequence the nanoprobes merge into a cluster, changing the light absorption.
Campos, Elida G; Jesuino, Rosália Santos Amorim; Dantas, Alessandra da Silva; Brígido, Marcelo de Macedo; Felipe, Maria Sueli S
Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.
ARANTES, Thales Domingos; BAGAGLI, Eduardo; NIÑO-VEGA, Gustavo; SAN-BLAS, Gioconda; THEODORO, Raquel Cordeiro
SUMMARY To commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field. PMID:26465366
Sandhu, G S; Aleff, R A; Kline, B C; da Silva Lacaz, C
Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi. PMID:9196219
Urán, Martha E.; Nosanchuk, Joshua D.; Restrepo, Angela; Hamilton, Andrew J.; Gómez, Beatriz L.; Cano, Luz E.
Several cell wall constituents, including melanins or melanin-like compounds, have been implicated in the pathogenesis of a wide variety of microbial diseases caused by diverse species of pathogenic bacteria, fungi, and helminthes. Among these microorganisms, the dimorphic fungal pathogen Paracoccidioides brasiliensis produces melanin in its conidial and yeast forms. In the present study, melanin particles from P. brasiliensis were injected into BALB/c mice in order to produce monoclonal antibodies (MAbs). We identified five immunoglobulin G1 (IgG1) κ-chain and four IgM melanin-binding MAbs. The five IgG1 κ-chain isotypes are the first melanin-binding IgG MAbs ever reported. The nine MAbs labeled P. brasiliensis conidia and yeast cells both in vitro and in pulmonary tissues. The MAbs cross-reacted with melanin-like purified particles from other fungi and also with commercial melanins, such as synthetic and Sepia officinalis melanin. Melanization during paracoccidioidomycosis (PCM) was also further supported by the detection of IgG antibodies reactive to melanin from P. brasiliensis conidia and yeast in sera and bronchoalveolar lavage fluids from P. brasiliensis-infected mice, as well as in sera from human patients with PCM. Serum specimens from patients with other mycoses were also tested for melanin-binding antibodies by enzyme-linked immunosorbent assay, and cross-reactivities were detected for melanin particles from different fungal sources. These results suggest that melanin from P. brasiliensis is an immunologically active fungal structure that activates a strong IgG humoral response in humans and mice. PMID:21813659
Lopera, Damaris; Urán-Jiménez, Martha Eugenia
Neutrophils predominate during the acute phase of the Paracoccidioides brasiliensis infection. Herein, we determined the role of the neutrophil during the early stages of experimental pulmonary paracoccidioidomycosis using a monoclonal antibody (mAb) specific for neutrophils. Male BALB/c mice were inoculated intranasally with 1.5 × 106 or 2 × 106 P. brasiliensis yeast cells. The mAb was administered 24 h before infection, followed by doses every 48 h until mice were sacrificed. Survival time was evaluated and mice were sacrificed at 48 h and 96 h after inoculation to assess cellularity, fungal load, cytokine/chemokine levels, and histopathological analysis. Neutrophils from mAb-treated mice were efficiently depleted (99.04%). Eighty percent of the mice treated with the mAb and infected with 1.5 × 106 yeast cells died during the first two weeks after infection. When mice were treated and infected with 2 × 106 yeast cells, 100% of them succumbed by the first week after infection. During the acute inflammatory response significant increases in numbers of eosinophils, fungal load and levels of proinflammatory cytokines/chemokines were observed in the mAb-treated mice. We also confirmed that neutrophils are an important source of IFN-γ and IL-17. These results indicate that neutrophils are essential for protection as well as being important for regulating the early inflammatory immune response in experimental pulmonary paracoccidioidomycosis. PMID:27642235
Vilela, Raquel; Bossart, Gregory D.; St. Leger, Judy A.; Dalton, Leslie M.; Reif, John S.; Schaefer, Adam M.; McCarthy, Peter J.; Fair, Patricia A.
Cutaneous granulomas in dolphins were believed to be caused by Lacazia loboi, which also causes a similar disease in humans. This hypothesis was recently challenged by reports that fungal DNA sequences from dolphins grouped this pathogen with Paracoccidioides brasiliensis. We conducted phylogenetic analysis of fungi from 6 bottlenose dolphins (Tursiops truncatus) with cutaneous granulomas and chains of yeast cells in infected tissues. Kex gene sequences of P. brasiliensis from dolphins showed 100% homology with sequences from cultivated P. brasiliensis, 73% with those of L. loboi, and 93% with those of P. lutzii. Parsimony analysis placed DNA sequences from dolphins within a cluster with human P. brasiliensis strains. This cluster was the sister taxon to P. lutzii and L. loboi. Our molecular data support previous findings and suggest that a novel uncultivated strain of P. brasiliensis restricted to cutaneous lesions in dolphins is probably the cause of lacaziosis/lobomycosis, herein referred to as paracoccidioidomycosis ceti. PMID:27869614
Mansano, Elaine Sciuniti Benites; de Morais, Gutierrez Rodrigues; Moratto, Edilaine Martins; Sato, Francielle; Medina Neto, Antonio; Svidzinski, Terezinha Ines Estivalet; Baesso, Mauro Luciano; Hernandes, Luzmarina
Paracoccidioidomycosis is the most important systemic mycosis in Latin America. The main entrance of the fungus is the airway. It primarily occurs in the lung, but in its disseminated form may affect any organ. The liver is one of the organs afflicted by this disease and its homeostasis may be impaired. The aim of the present study was to evaluate the evolution of paracoccidioidomycosis in the liver of Swiss mice and correlate morphological factors with the expression of gp43 and with physicochemical analysis via FT-Raman of the infected organ. According to colony forming unit (CFU) and granuloma counting, the first and second weeks were the periods when infection was most severe. Tissue response was characterized by the development of organized granulomas and widespread infection, with yeasts located within the macrophages and isolated hepatocytes. The gp43 molecule was distributed throughout the hepatic parenchyma, and immunostaining was constant in all observed periods. The main physicochemical changes of the infected liver were observed in the spectral ranges between 1700–1530 cm−1 and 1370 – 1290 cm−1, a peak shifting center attributed to phenylalanine and area variation of -CH2 and -CH3 compounds associated to collagen, respectively. Over time, there was a direct proportional relationship between the number of CFUs, the number of granulomas and the physicochemical changes in the liver of mice infected with Paracoccidioides brasiliensis. The expression of gp43 was similar in all observed periods. PMID:25181524
Scorzoni, Liliana; de Paula e Silva, Ana Carolina Alves; Singulani, Junya de Lacorte; Leite, Fernanda Sangalli; de Oliveira, Haroldo Cesar; da Silva, Rosangela Aparecida Moraes; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares
Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction.
Scorzoni, Liliana; de Paula e Silva, Ana Carolina Alves; Singulani, Junya de Lacorte; Leite, Fernanda Sangalli; de Oliveira, Haroldo Cesar; Moraes da Silva, Rosangela Aparecida; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares
Paracoccidioidomycosis is a systemic mycosis, endemic in Latin America. The etiologic agents of this mycosis are composed of 2 species: Paracoccidioides brasiliensis and P. lutzii. Murine animal models are the gold standard for in vivo studies; however, ethical, economical and logistical considerations limit their use. Galleria mellonella is a suitable model for in vivo studies of fungal infections. In this study, we compared the virulence of P. brasiliensis and P. lutzii in G. mellonella model. The deaths of larvae infected with P. brasiliensis or P. lutzii were similar, and both species were able to reduce the number of hemocytes, which were estimated by microscopy and flow cytometer. Additionally, the phagocytosis percentage was similar for both species, but when we analyze hemocyte-Paracoccidioides spp. interaction using flow cytometer, P. lutzii showed higher interactions with hemocytes. The gene expression of gp43 as well as this protein was higher for P. lutzii, and this expression may contribute to a greater adherence to hemocytes. These results helped us evaluate the behavior of Paracoccidioides spp in G. mellonella, which is a convenient model for investigating the host-Paracoccidioides spp. interaction. PMID:26552324
Alegre, Ana Claudia Paiva; Oliveira, Aline Ferreira; Dos Reis Almeida, Fausto Bruno; Roque-Barreira, Maria Cristina; Hanna, Ebert Seixas
Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The
McEwen, J G; Restrepo, B I; Salazar, M E; Restrepo, A
More than 80% of the conidia produced by two different isolates of Paracoccidioides brasiliensis, were found to be uninucleate; however, when they were incubated at 37 degrees C and began to transform into yeast cells, they became bi- or multi-nucleated, so that when most of the conidia had already transformed into yeast cells (72-96 h), there were at least four or five nuclei per cell in approximately 80% of the conidia examined.
Andrade, Rosângela V; Da Silva, Silvana P; Torres, Fernando A G; Poças-Fonseca, Marcio José; Silva-Pereira, Ildenete; Maranhão, Andrea Q; Campos, Elida G; Moraes, Lídia Maria P; Jesuíno, Rosália S A; Pereira, Maristela; Soares, Célia M A; Walter, Maria Emília M T; Carvalho, Maria Joseá A; Almeida, Nalvo F; Brigido, Marcelo M; Felipe, Maria Sueli S
Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.
Alves da Costa, Thiago; Di Gangi, Rosária; Martins, Paula; Longhini, Ana Leda Figueiredo; Zanucoli, Fábio; de Oliveira, Alexandre Leite Rodrigues; Stach-Machado, Dagmar Ruth; Burger, Eva; Verinaud, Liana; Thomé, Rodolfo
Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8+ T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8+ T cells being the main source. Blockage of cross-presentation to CD8+ T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis. PMID:26302057
Burger, E; Vaz, C C; Sano, A; Calich, V L; Singer-Vermes, L M; Xidieh, C F; Kashino, S S; Nishimura, K; Miyaji, M
Athymic and euthymic BALB/c mice infected with highly (Pb18) or slightly (Pb265) virulent Paracoccidioides brasiliensis isolates were compared regarding mortality, presence of viable yeasts, specific immunoglobulin M (IgM) and IgG titers, and the antigen recognition patterns of these antibodies. Isolate Pb18 caused a more severe disease in athymic mice, as supported by higher number of infected organs and shorter survival times. These animals, however, were resistant to Pb265 infection. High titers of antibodies were found only in euthymic mice, seven weeks after Pb18 infection. At this time, euthymic animals presented IgG antibodies to numerous protein bands that were not detected at four weeks postinfection or after Pb265 inoculation. In contrast, antibodies from athymic mice always reacted with few antigen bands. Although the majority of P. brasiliensis antigens are T cell-dependent, the immunodominant gp43 and also the 41.5- and 27.5-kD antigens are here, for the first time, characterized as T cell-independent antigens of P. brasiliensis.
Arraes, Fabrício B M; Benoliel, Bruno; Burtet, Rafael T; Costa, Patrícia L N; Galdino, Alexandro S; Lima, Luanne H A; Marinho-Silva, Camila; Oliveira-Pereira, Luciana; Pfrimer, Pollyanna; Procópio-Silva, Luciano; Reis, Viviane Castelo-Branco; Felipe, Maria Sueli S
Annotation of the transcriptome of the dimorphic fungus Paracoccidioides brasiliensis has set the grounds for a global understanding of its metabolism in both mycelium and yeast forms. This fungus is able to use the main carbohydrate sources, including starch, and it can store reduced carbons in the form of glycogen and trehalose; these provide energy reserves that are relevant for metabolic adaptation, protection against stress and infectivity mechanisms. The glyoxylate cycle, which is also involved in pathogenicity, is present in this fungus. Classical pathways of lipid biosynthesis and degradation, including those of ketone body and sterol production, are well represented in the database of P. brasiliensis. It is able to synthesize de novo all nucleotides and amino acids, with the sole exception of asparagine, which was confirmed by the fungus growth in minimal medium. Sulfur metabolism, as well as the accessory synthetic pathways of vitamins and co-factors, are likely to exist in this fungus.
Felipe, Maria Sueli S; Torres, Fernando A G; Maranhão, Andrea Q; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio J; Campos, Elida G; Moraes, Lídia M P; Arraes, Fabrício B M; Carvalho, Maria José A; Andrade, Rosângela V; Nicola, André M; Teixeira, Marcus M; Jesuíno, Rosália S A; Pereira, Maristela; Soares, Célia M A; Brígido, Marcelo M
Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.
Bonilla-Abadía, F.; Vélez, J. D.; Zárate-Correa, L. C.; Carrascal, E.; Guarín, N.; Castañeda-Ramírez, C. R.; Cañas, C. A.
Paracoccidioidomycosis is an endemic South American systemic mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). The main clinical form of disease is pulmonary, but all organs may be involved. We report a case of overinfection by P. brasiliensis in chronic gouty arthritis affecting the proximal phalanx of the right hallux. The patient required proximal amputation and long-term antifungal therapy. PMID:23251162
Carbonell, Luis M.
The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M ⇌ Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M → Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y → M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M → Y, and intrayeast hyphae during the Y → M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon. Images PMID:5359610
Gago, J; Godio, C; Ochoa, L; Negroni, R; Nejamkis, M R
The goal of this work was to develop in solid medium a fast method to obtain Paracoccidioides brasiliensis (Pb) with a high yield. Four culture media were assayed: Sabouraud honey-agar, Sabouraud dextrose-agar, tomato -agar-medium (TOM) and a medium based on grape pulp. The most exhuberant growth was observed in medium based on grape pulp. Antigen was prepared in microscale at 6, 10 and 15 days incubation of solid cultures and the crude product concentrated by means of Centriplus tubes (Helena, France). Isolated antigens were subjected to polyacrylamide gel electrophoresis, followed by immunolabelling and detection of the characteristic gp45 antigen employing human and Pb-infected rat sera. Best results were observed after 10 days culture in grape medium. None of the other three media afforded comparable results.
de Araújo, Eliseu Frank; Medeiros, Daniella Helena; Condino-Neto, Antônio
Plasmacytoid dendritic cells (pDCs), considered critical for immunity against viruses, were recently associated with defense mechanisms against fungal infections. However, the immunomodulatory function of pDCs in pulmonary paracoccidiodomycosis (PCM), an endemic fungal infection of Latin America, has been poorly defined. Here, we investigated the role of pDCs in the pathogenesis of PCM caused by the infection of 129Sv mice with 1 x 106 P. brasiliensis-yeasts. In vitro experiments showed that P. brasiliensis infection induces the maturation of pDCs and elevated synthesis of TNF-α and IFN-β. The in vivo infection caused a significant influx of pDCs to the lungs and increased levels of pulmonary type I IFN. Depletion of pDCs by a specific monoclonal antibody resulted in a less severe infection, reduced tissue pathology and increased survival time of infected mice. An increased influx of macrophages and neutrophils and elevated presence of CD4+ and CD8+ T lymphocytes expressing IFN-γ and IL-17 in the lungs of pDC-depleted mice were also observed. These findings were concomitant with decreased frequency of Treg cells and reduced levels of immunoregulatory cytokines such as IL-10, TGF-β, IL-27 and IL-35. Importantly, P. brasilienis infection increased the numbers of pulmonary pDCs expressing indoleamine 2,3-dioxygenase-1 (IDO), an enzyme with immunoregulatory properties, that were reduced following pDC depletion. In agreement, an increased immunogenic activity of infected pDCs was observed when IDO-deficient or IDO-inhibited pDCs were employed in co-cultures with lymphocytes Altogether, our results suggest that in pulmonary PCM pDCs exert a tolerogenic function by an IDO-mediated mechanism that increases Treg activity. PMID:27992577
da Silva, Thiago Aparecido; Roque-Barreira, Maria Cristina; Casadevall, Arturo; Almeida, Fausto
Extracellular vesicles (EVs) released by eukaryotes, archaea, and bacteria contain proteins, lipids, polysaccharides, and other molecules. The cargo analysis of EVs shows that they contain virulence factors suggesting a role in the pathogenesis of infection. The proteome, lipidome, RNA content, and carbohydrate composition of EVs from Paracoccidioides brasiliensis and Paracoccidioides lutzii were characterized. However, the effects of P. brasiliensis EVs on the host immune system have not yet been investigated. Herein, we verified that EVs from P. brasiliensis induce the production of proinflammatory mediators by murine macrophages in a dose-dependent manner. Addition of EV to macrophages also promoted transcription of the M1-polarization marker iNOs and diminish that of the M2 markers Arginase-1, Ym-1, and FIZZ-1. Furthermore, the augmented expression of M2-polarization markers, stimulated by IL-4 plus IL-10, was reverted toward an M1 phenotype in response to secondary stimulation with EVs from P. brasiliensis. The ability of EVs from P. brasiliensis to promote M1 polarization macrophages favoring an enhanced fungicidal activity, demonstrated by the decreased CFU recovery of internalized yeasts, with comparable phagocytic efficacy. Our results suggest that EVs from P. brasiliensis can modulate the innate immune response and affect the relationship between P. brasiliensis and host immune cells. PMID:27775058
de Souza Silva, Calliandra; Tavares, Aldo Henrique; Sousa Jeronimo, Marcio; Soares de Lima, Yasmin; da Silveira Derengowski, Lorena; Lorenzetti Bocca, Anamélia; Silva-Pereira, Ildinete
Considering the importance of macrophages as the first line of defense against fungal infection and the different roles played by the two M1- and M2-like polarized macrophages, we decided to evaluate the effects of Paracoccidioides brasiliensis infection on GM-CSF- and M-CSF-induced bone marrow-derived macrophages (BMM) from the A/J and B10.A mouse strains, an established model of resistance/susceptibility to PCM, respectively. Upon differentiation, the generated GM- or M-BMMs were characterized by morphological analyses, gene expression profiles, and cytokines production. Our main results demonstrate that GM-BMMs derived from A/J and B.10 produced high levels of pro- and anti-inflammatory cytokines that may contribute to generate an unbalanced early immune response. In accordance with the literature, the B10.A susceptible mice lineage has an innate tendency to polarize into M1-like phenotype, whereas the opposite phenotype occurs in A/J resistance mice. In this context, our data support that susceptibility and resistance are strongly correlated with M1 and M2 polarization, respectively. PMID:26543326
de Souza Silva, Calliandra; Tavares, Aldo Henrique; Sousa Jeronimo, Marcio; Soares de Lima, Yasmin; da Silveira Derengowski, Lorena; Bocca, Anamélia Lorenzetti; Silva-Pereira, Ildinete
Considering the importance of macrophages as the first line of defense against fungal infection and the different roles played by the two M1- and M2-like polarized macrophages, we decided to evaluate the effects of Paracoccidioides brasiliensis infection on GM-CSF- and M-CSF-induced bone marrow-derived macrophages (BMM) from the A/J and B10.A mouse strains, an established model of resistance/susceptibility to PCM, respectively. Upon differentiation, the generated GM- or M-BMMs were characterized by morphological analyses, gene expression profiles, and cytokines production. Our main results demonstrate that GM-BMMs derived from A/J and B.10 produced high levels of pro- and anti-inflammatory cytokines that may contribute to generate an unbalanced early immune response. In accordance with the literature, the B10.A susceptible mice lineage has an innate tendency to polarize into M1-like phenotype, whereas the opposite phenotype occurs in A/J resistance mice. In this context, our data support that susceptibility and resistance are strongly correlated with M1 and M2 polarization, respectively.
Calich, V. L.; Singer-Vermes, L. M.; Siqueira, A. M.; Burger, E.
Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus. PMID:4063162
Arango, Julián Camilo
Chronic stages of paracoccidioidomycosis (PCM) are characterized by granulomatous lesions which promote the development of pulmonary fibrosis leading to the loss of respiratory function in 50% of patients; in addition, it has been observed that neutrophils predominate during these chronic stages of P. brasiliensis infection. The goal of this study was to evaluate the role of the neutrophil during the chronic stages of experimental pulmonary PCM and during the fibrosis development and tissue repair using a monoclonal specific to this phagocytic cell. Male BALB/c mice were inoculated intranasally with 1.5x106 P. brasiliensis yeast cells. A monoclonal antibody specific to neutrophils was administered at 4 weeks post-inoculation followed by doses every 48h during two weeks. Mice were sacrificed at 8 and 12 weeks post-inoculation to assess cellularity, fungal load, cytokine/chemokine levels, histopathological analysis, collagen and expression of genes related to fibrosis development. Depletion of neutrophils was associated with a significant decrease in the number of eosinophils, dendritic cells, B cells, CD4-T cells, MDSCs and Treg cells, fungal load and levels of most of the pro-inflammatory cytokines/chemokines evaluated, including IL-17, TNF-α and TGF-β1. Recovery of lung architecture was also associated with reduced levels of collagen, high expression of TGF-β3, matrix metalloproteinase (MMP)-12 and -14, and decreased expression of tissue inhibitor metalloproteinase (TIMP)-2, and MMP-8. Depletion of neutrophils might attenuate lung fibrosis and inflammation through down-regulating TGF-β1, TNF-α, IL-17, MMP-8 and TIMP-2. These results suggest that neutrophil could be considered as a therapeutic target in pulmonary fibrosis induced by P. brasiliensis. PMID:27690127
Benoliel, Bruno; Arraes, Fabrício B M; Reis, Viviane Castelo-Branco; Siqueira, Saulo J L de; Parachin, Nádia S; Torres, Fernando A G
Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.
Loth, Eduardo Alexandre; Biazin, Samia Khalil; Paula, Claudete Rodrigues; Simão, Rita de Cássia Garcia; de Franco, Marcello Fabiano; Puccia, Rosana; Gandra, Rinaldo Ferreira
Paracoccidioidomycosis (PCM), a disease caused by the fungus Paracoccidioides brasiliensis (Pb), is highly prevalent in Brazil, where it is the principal cause of death by systemic mycoses. The disease primarily affects men aged 30-50 year old and usually starts as a pulmonary focus and then may spread to other organs and systems, including the joints. The present study aimed to develop an experimental model of paracoccidioidomycotic arthritis. Two-month-old male Wistar rats (n = 48) were used, divided in 6 groups: test groups EG/15 and EG/45 (received one dose of 100 μl of saline containing 10(5) Pb viable yeasts in the knee); heat killed Pb-group HK/15 and HK/45 (received a suspension of 10(5) Pb nonviable yeasts in the knee) and control groups CG/15 and CG/45 (received only sterile saline in the knee). The rats were killed 15 and 45 days postinoculation. In contrast with the control rats, the histopathology of the joints of rats of the test groups (EG/15 and EG/45) revealed a picture of well-established PCM arthritis characterized by extensive sclerosing granulomatous inflammation with numerous multiple budding fungal cells. The X-ray examination revealed joint alterations in these groups. Only metabolic active fungi evoked inflammation. The experimental model was able to induce fungal arthritis in the knees of the rats infected with metabolic active P. brasiliensis. The disease tended to be regressive and restrained by the immune system. No evidence of fungal dissemination to the lungs was observed.
Antigenic components of the yeast extract of Paracoccidioides brasiliensis Linder 2511 cultured for 3, 8, 20, 30, and 60 days were examined by the Western blot (immunoblot) technique. The 3-day extract was chosen for characterization of the antigenic components because its stability did not vary with time and it contained all antigens identified by patient sera. Antibodies to cross-reacting antigens of P. brasiliensis extracts were detected in sera from patients with histoplasmosis, candidiasis, and aspergillosis. The 58-, 57-, 21-, and 16-kilodalton (kDa) antigens were specific for P. brasiliensis, while the 48- and 45-kDa antigens were specific for paracoccidioidomycosis. The Western blot technique is a useful tool for the diagnosis of disease and revealed heterogeneity in the responses of patient sera. The combination of the 58-, 57-, and 45-kDa proteins confirmed a diagnosis of paracoccidioidomycosis (87% of the cases). Images PMID:2380351
de Lacorte Singulani, Junya; Scorzoni, Liliana; de Paula E Silva, Ana Carolina Alves; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares
Paracoccidioides brasiliensis and P. lutzii belong to a group of thermodimorphic fungi and cause paracoccidioidomycosis (PCM), which is a human systemic mycosis endemic in South and Central America. Patients with this mycosis are commonly treated with amphotericin B (AmB) and azoles. The study of fungal virulence and the efficacy and toxicity of antifungal drugs has been successfully performed in a Galleria mellonella infection model. In this work, G. mellonella larvae were infected with two Paracoccidioides spp. and the efficacy and toxicity of AmB and itraconazole were evaluated in this model for the first time. AmB and itraconazole treatments were effective in increasing larval survival and reducing the fungal burden. The fungicidal and fungistatic effects of AmB and itraconazole, respectively, were observed in the model. Furthermore, these effects were independent of changes in haemocyte number. G. mellonella can serve as a rapid model for the screening of new antifungal compounds against Paracoccidioides and can contribute to a reduction in experimental animal numbers in the study of PCM.
Cunha, Daniela A.; Zancopé-Oliveira, Roseli M.; Sueli, M.; Felipe, S.; Salem-Izacc, Silvia M.; Deepe Jr., George S.; Soares, Célia M. A.
The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients. PMID:11874881
Mejía, Susana P; Cano, Luz E; López, Juan A; Hernandez, Orville; González, Ángel
Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.
Sardi, Janaina de Cássia Orlandi; Pitangui, Nayla de Souza; Voltan, Aline Raquel; Braz, Jaqueline Derissi; Machado, Marcelo Pelajo; Fusco Almeida, Ana Marisa; Mendes Giannini, Maria Jose Soares
Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 μm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment. PMID:26055497
Restrepo, A; Arango, M D
A total of 60 clinical isolates of Paracoccidioides brasiliensis were tested for susceptibility to sulfadiazine and sulfadimethoxyne by the agar dilution technique. A modification of the Mueller-Hinton medium was devised which gave good growth of the yeast form. The minimum inhibitory concentrations for only 51.6% of the isolates were in the range of the recommended blood serum concentration (50 micrograms/ml). For 6 to 8% of the isolates, the minimum inhibitory concentrations were above 200 micrograms of both sulfadiazine and sulfadimethoxyne per ml. A significant decreases in susceptibility was demonstrated for one isolate obtained from a patient relapsing during sulfonamide therapy. Images PMID:7416744
Dantas, Alessandra S; Andrade, Rosângela V; de Carvalho, Maria J; Felipe, Maria Sueli S; Campos, Elida G
Paracoccidioides brasiliensis is a dimorphic fungus that infects humans and establishes infection in the yeast form. We are interested in the mechanisms this fungus uses to evade the human immune system, and in its survival strategies within infected host cells. Reactive oxygen species play an important role in host defence, but are detoxified by pathogen-derived antioxidant enzymes to prevent oxidative damage. The transcriptional and post-transcriptional regulation of P. brasiliensis catalase and cytochrome-c peroxidase (CCP) antioxidant enzymes upon culture treatment with hydrogen peroxide (H(2)O(2)) is described. High H(2)O(2) concentrations (up to 100 mm) still permitted 70-100% survival of exponential and stationary phase yeast cells, though stationary phase cells were consistently more resistant. P. brasiliensis has both cytosolic and peroxisomal catalase isoenzymes and a single cytochrome-c peroxidase. High-dose treatments with H(2)O(2) led to an early increase in total catalase and CCP enzymatic activities, indicative of post-transcriptional regulation. The expression levels of the catalase genes increased three to fourfold when the cells were treated with 50 mm H(2)O(2) for 40 or 50 min. Lipid peroxidation, as assessed by the thiobarbituric acid method, was relatively low upon treatment with H(2)O(2), which was consistent with our results demonstrating that P. brasiliensis has a powerful antioxidant defence system enabling it to survive H(2)O(2)-mediated stress.
Cano, L E; Arango, R; Salazar, M E; Brummer, E; Stevens, D A; Restrepo, A
The ability of conidia, the infectious form of the dimorphic fungus Paracoccidioides brasiliensis, to be killed in vitro by murine pulmonary macrophages was studied. Mice were immunized by intravenous injection of killed conidia, which resulted in cellular immunity demonstrated by delayed type hypersensitivity in vivo and macrophage migration inhibition factor production in vitro. Resident pulmonary macrophages from non-immune mice were able to significantly kill the conidia (28%). Such macrophages treated with supernatants (cytokines) from antigen-stimulated immune mononuclears had a markedly enhanced ability to kill conidia (73%). These results show that activated pulmonary macrophages are potent killers of conidia of P. brasiliensis and that immune mononuclears play a role in activation of macrophages. Activated macrophages may be important for pulmonary defense against the initial stages of infection with this fungus.
Finquelievich, J L; Negroni, R; Iovannitti, C A; de Elías Costa, M R
A comparative study of antigenicity and pathogenicity for rats of six Paracoccidioides brasiliensis strains was carried out. The antigenic capacity "in vitro" of cytoplasmic extract from each strain was determined by immunodiffusion test against 6 serum samples obtained from rats experimentally infected with Paracoccidioides brasiliensis, that had presented positive reactions with a metabolic control antigen. The cytoplasmic extracts were used at final concentration of 100 mg/ml. All of them showed 2 or 3 precipitation bands in this assay. One hundred twenty Wistar rats both sexes weighing approximately 200 g, were inoculated intracardiacally with suspensions of the yeast phase of different P. brasiliensis strains. Two concentrations containing 3 x 10(7) and 5 x 10(7) cells/ml of each isolate were prepared. The inoculated animals were divided in two groups, one was left to its spontaneous outcome and the percentages of deaths were registered and the other rats were sacrificed at 14, 28, 56 and 70 days post-infection. The following parameters were taken into account for evaluation: A) presence of macroscopic granulomas in lung, liver, spleen and kidney; B) presence of P. brasiliensis in microscopic exams of the same organs, in wet preparations and in histologic sections stained by H&E; C) culture of lung and D) immunodiffusion test using pre-mortem serum samples and the homologous antigen. The correlation between the most important parameters studied in each strain are summarized as follow: As no significant differences between the two inocula employed for each strain was observed, the before mention results are the average of those obtained with each inoculation doses.(ABSTRACT TRUNCATED AT 250 WORDS)
Machado, Gabriel Capella; Moris, Daniela Vanessa; Arantes, Thales Domingos; Silva, Luciane Regina Franciscone; Theodoro, Raquel Cordeiro; Mendes, Rinaldo Pôncio; Vicentini, Adriana Pardini; Bagagli, Eduardo
We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens. PMID:23903981
Costa, Christiane da Silva; Albuquerque, Flávia Caixeta; Andrade, Rosângela Vieira; Oliveira, Gina Camilo de; Almeida, Mauro Fernandes de; Brigido, Marcelo de Macedo; Maranhão, Andrea Queiroz
In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.
Menino, João Filipe; Saraiva, Margarida; Gomes-Alves, Ana G.; Lobo-Silva, Diogo; Sturme, Mark; Gomes-Rezende, Jéssica; Saraiva, Ana Laura; Goldman, Gustavo H.; Cunha, Cristina; Carvalho, Agostinho; Romani, Luigina; Pedrosa, Jorge; Castro, António Gil; Rodrigues, Fernando
Background Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear. Methodology/Principal findings We used in vitro and in vivo models of infection by P. brasiliensis, comparing wild type and TLR9 deficient (−/−) mice, to assess the contribution of TLR9 on cytokine induction, phagocytosis and outcome of infection. We show that TLR9 recognizes either the yeast form or DNA from P. brasiliensis by stimulating the expression/production of pro-inflammatory cytokines by bone marrow derived macrophages, also increasing their phagocytic ability. We further show that TLR9 plays a protective role early after intravenous infection with P. brasiliensis, as infected TLR9−/− mice died at higher rate during the first 48 hours post infection than wild type mice. Moreover, TLR9−/− mice presented tissue damage and increased expression of several cytokines, such as TNF-α and IL-6. The increased pattern of cytokine expression was also observed during intraperitoneal infection of TLR9−/− mice, with enhanced recruitment of neutrophils. The phenotype of TLR9−/− hosts observed during the early stages of P. brasiliensis infection was reverted upon a transient, 48 hours post-infection, neutrophil depletion. Conclusions/Significance Our results suggest that TLR9 activation plays an early protective role against P. brasiliensis, by avoiding a deregulated type of inflammatory response associated to neutrophils that may lead to tissue damage. Thus
Albano, A P N; Klafke, G B; Brandolt, T M; Da Hora, V P; Minello, L F; Jorge, S; Santos, E O; Behling, G M; Camargo, Z P; Xavier, M O; Meireles, M C A
Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20%), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.
de Freitas, Roseli Santos; Dantas, Kátia Cristina; Garcia, Roberta Scholz Pinto; Magri, Marcello Mihailenko Chaves; de Andrade, Heitor Franco
Paracoccidioidomycosis is a systemic mycosis with a geographic distribution that is limited to Central and South America; Brazil has the highest number of cases. Severe disseminated disease caused by paracoccidioidomycosis was observed in acquired immunodeficiency syndrome patients who live or have resided in endemic paracoccidioidomycosis areas. Here we describe a male patient admitted to a large public hospital with diffuse nodular infiltrates observed in chest radiographs and with erosion at the second rib near the sternum. Blood tests showed anti-human immunodeficiency virus antibodies, a human immunodeficiency virus viral load of 59,700 (4.8 log), and CD4 144/mm(3), with negative serology result for fungal infections. Aspirate of the rib lesion showed cells with a typical morphology of Paracoccidioides brasiliensis, aside from benign inflammatory cells. The histology of the rib biopsy showed typical granulomas and immunostained fungal cells. Although there was no growth in the Sabouraud cultures, Paracoccidioides brasiliensis gp43 and rDNA genes were detected in the aspirate by polymerase chain reaction. Therapy with amphotericin resulted in complete recovery. This type of bone lesion is rare and has been described primarily in the juvenile form of paracoccidioidomycosis; it must be included in the differential diagnosis of bone lesions in adult acquired immunodeficiency syndrome patients of endemic areas.
Parente, Ana F. A.; Bailão, Alexandre M.; Borges, Clayton L.; Parente, Juliana A.; Magalhães, Adriana D.; Ricart, Carlos A. O.; Soares, Célia M. A.
Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM). The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D) gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways. PMID:21829521
Parente-Rocha, Juliana Alves; Parente, Ana Flávia Alves; Baeza, Lilian Cristiane; Bonfim, Sheyla Maria Rondon Caixeta; Hernandez, Orville; McEwen, Juan G.; Bailão, Alexandre Melo; Taborda, Carlos Pelleschi; Borges, Clayton Luiz; Soares, Célia Maria de Almeida
Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection. PMID:26360774
Parente-Rocha, Juliana Alves; Parente, Ana Flávia Alves; Baeza, Lilian Cristiane; Bonfim, Sheyla Maria Rondon Caixeta; Hernandez, Orville; McEwen, Juan G; Bailão, Alexandre Melo; Taborda, Carlos Pelleschi; Borges, Clayton Luiz; Soares, Célia Maria de Almeida
Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.
Carrero, Lilia L; Niño-Vega, Gustavo; Teixeira, Marcus M; Carvalho, Maria Jose A; Soares, Célia M A; Pereira, Maristela; Jesuino, Rosália S A; McEwen, Juan G; Mendoza, Leonel; Taylor, John W; Felipe, Maria Sueli; San-Blas, Gioconda
By means of genealogical concordance phylogenetic species recognition (GCPSR), we have investigated coding and non-coding regions from various genes and the ITS sequences of 7 new and 14 known isolates of Paracoccidioides brasiliensis. Such isolates grouped within the three phylogenetic groups recently reported in the genus Paracoccidioides, with one single exception, i.e., Pb01, a strain that has been the subject of intense molecular studies for many years. This isolate clearly separates from all other Paracoccidioides isolates in phylogenetic analyses and greatly increases the genomic variation known in this genus.
Cardoso, Maria Angélica G; Tambor, José Humberto M; Nobrega, Francisco G
We present here the sequence of the mitochondrial DNA of the pathogenic thermodimorphic fungus Paracoccidioides brasiliensis, agent of an endemic disease in most South American countries. The sequenced genome has 71 334 bp and is organized as a circular molecule with two gaps of unknown size flanking the middle exon of the nad5 gene. We located genes coding for the three subunits of the ATP synthase (atp6, atp8 and atp9), the apocytochrome b (cob), three subunits of the cytochrome c oxidase enzyme complex (cox1, cox2 and cox3), seven subunits of the reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase (nad1, nad2, nad3, nad4, nad5, nad6 and nad4L) and the large (rnl) and small (rns) subunits of ribosomal RNA. Two maturases and a ribosomal protein (rms5) are located inside introns. Twenty-five tRNAs were identified with acceptors for all 20 amino acids. Seven polypurine/polypyrimidine tracts (140-240 bp) have been found in this genome. All genes are in the same orientation over the genome, while their order is closest to the mitochondrial genomes from Penicillium marneffei and Aspergillus nidulans.
Moreira, Sabrina F I; Bailão, Alexandre M; Barbosa, Mônica S; Jesuino, Rosalia S A; Felipe, M Sueli Soares; Pereira, Maristela; de Almeida Soares, Célia Maria
Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.
Bonfim, Sheyla M R C; Cruz, Aline H S; Jesuino, Rosália S A; Ulhoa, Cirano J; Molinari-Madlum, Eugênia E W I; Soares, Célia M A; Pereira, Maristela
A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.
Cunha, A F; Sousa, M V; Silva, S P; Jesuíno, R S; Soares, C M; Felipe, M S
Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.
Travassos, Luiz R; Taborda, Carlos P
Dimorphic fungi are agents of systemic mycoses associated with significant morbidity and frequent lethality in the Americas. Among the pathogenic species are Paracoccidioides brasiliensis and Paracoccidioides lutzii, which predominate in South America; Histoplasma capsulatum, Coccidioides posadasii, and Coccidioides immitis, and the Sporothrix spp. complex are other important pathogens. Associated with dimorphic fungi other important infections are caused by yeast such as Candida spp. and Cryptococcus spp. or mold such as Aspergillus spp., which are also fungal agents of deadly infections. Nowadays, the actual tendency of therapy is the development of a pan-fungal vaccine. This is, however, not easy because of the complexity of eukaryotic cells and the particularities of different species and isolates. Albeit there are several experimental vaccines being studied, we will focus mainly on peptide vaccines or epitopes of T-cell receptors inducing protective fungal responses. These peptides can be carried by antibody inducing β-(1,3)-glucan oligo or polysaccharides, or be mixed with them for administration. The present review discusses the efficacy of linear peptide epitopes in the context of antifungal immunization and vaccine proposition.
Travassos, Luiz R.; Taborda, Carlos P.
Dimorphic fungi are agents of systemic mycoses associated with significant morbidity and frequent lethality in the Americas. Among the pathogenic species are Paracoccidioides brasiliensis and Paracoccidioides lutzii, which predominate in South America; Histoplasma capsulatum, Coccidioides posadasii, and Coccidioides immitis, and the Sporothrix spp. complex are other important pathogens. Associated with dimorphic fungi other important infections are caused by yeast such as Candida spp. and Cryptococcus spp. or mold such as Aspergillus spp., which are also fungal agents of deadly infections. Nowadays, the actual tendency of therapy is the development of a pan-fungal vaccine. This is, however, not easy because of the complexity of eukaryotic cells and the particularities of different species and isolates. Albeit there are several experimental vaccines being studied, we will focus mainly on peptide vaccines or epitopes of T-cell receptors inducing protective fungal responses. These peptides can be carried by antibody inducing β-(1,3)-glucan oligo or polysaccharides, or be mixed with them for administration. The present review discusses the efficacy of linear peptide epitopes in the context of antifungal immunization and vaccine proposition. PMID:28344577
Munk, M E; Kaufmann, S H
Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308
Restrepo, A; Cano, L E; de Bedout, C; Brummer, E; Stevens, D A
The viability of Paracoccidioides brasiliensis yeast-form cells was determined by colony-forming units, direct fluorescent staining, and production of germ tubes in slide culture. The first procedure was unreliable and time consuming; the latter two showed better correlation with hemacytometer total cell counts and required significantly less time. PMID:7107858
San-Blas, G; San-Blas, F; Gil, F; Mariño, L; Apitz-Castro, R
Ajoene, a garlic-derived compound that prevents platelet activation, inhibited the growth of Paracoccidioides brasiliensis, a fungal pathogen for humans, by affecting the integrity of the fungal cytoplasmic membrane. This action may be the basis for the study of ajoene as a possible specific antifungal drug. Images PMID:2817865
Felipe, M S S; Andrade, R V; Petrofeza, S S; Maranhão, A Q; Torres, F A G; Albuquerque, P; Arraes, F B M; Arruda, M; Azevedo, M O; Baptista, A J; Bataus, L A M; Borges, C L; Campos, E G; Cruz, M R; Daher, B S; Dantas, A; Ferreira, M A S V; Ghil, G V; Jesuino, R S A; Kyaw, C M; Leitão, L; Martins, C R; Moraes, L M P; Neves, E O; Nicola, A M; Alves, E S; Parente, J A; Pereira, M; Poças-Fonseca, M J; Resende, R; Ribeiro, B M; Saldanha, R R; Santos, S C; Silva-Pereira, I; Silva, M A S; Silveira, E; Simões, I C; Soares, R B A; Souza, D P; De-Souza, M T; Andrade, E V; Xavier, M A S; Veiga, H P; Venancio, E J; Carvalho, M J A; Oliveira, A G; Inoue, M K; Almeida, N F; Walter, M E M T; Soares, C M A; Brígido, M M
Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.
Silva, W P; Soares, R B; Jesuino, R S; Izacc, S M; Felipe, M S; Soares, C M
In this study we analyzed the expression of (alpha-tubulin during the dimorphic transition of the human-pathogenic fungus Paracoccidioides brasiliensis. The alpha-tubulin from P. brasiliensis was recognized by a commercially available anti-tubulin antibody and was developmentally regulated during the dimorphic form transition. We detected at least two alpha-tubulin isoforms in the mycelial state and only one isoform in the yeast forms. This finding suggests specific roles for the alpha-tubulin isoforms in P. brasiliensis's yeast and mycelial forms.
Loth, Eduardo Alexandre; Biazim, Samia Khalil; Dos Santos, José Henrique Fermino Ferreira; Puccia, Rosana; Brancalhão, Rosimeire Costa; Chasco, Lucinéia de Fátima; Gandra, Rinaldo Ferreira; Simão, Rita de Cássia Garcia; de Franco, Marcello Fabiano
Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.
Mendes, Josiara F; Klafke, Gabriel B; Albano, Ana Paula N; Cabana, Ângela L; Teles, Alessandra J; de Camargo, Zoilo P; Xavier, Melissa O; Meireles, Mário Carlos A
Paracoccidioidomycosis (PCM) is a systemic mycosis that occurs in several Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus Paracoccidioides spp. Serological studies to detect animal infection represent an excellent strategy for data on the agent's ecology. Although the state of Rio Grande do Sul (RS) is an endemic area for PCM in humans, there is scarce information available on the ecology of the agent in the region. This study aimed to investigate the infection by Paracoccidioides lutzii in animals living in RS, Brazil. A total of 85 wild mammals, 200 horses and 196 domestic dogs, previously tested for infection by P. brasiliensis, were included in this study. Serum samples from the animals were tested by ELISA to detect anti- P. lutzii antibodies. From the 481 animals tested, 105 (21.8%) were seropositive for IgG anti-P. lutzii. Of these, 54 were also positive for P. brasiliensis. A total of 11 horses (10.5%), 30 dogs (28.8%) and 10 wild mammals (9.5%) were positive only for P. lutzii (n=51). The detection of anti-P. lutzii antibodies in animals of RS suggests that the fungus can be found in southern Brazil, despite being described mainly in the midwest and southeast of the country.
Arantes, Thales Domingos; Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Bagagli, Eduardo
BACKGROUND Fluorescence in situ hybridisation (FISH) associated with Tyramide Signal Amplification (TSA) using oligonucleotides labeled with non-radioactive fluorophores is a promising technique for detection and differentiation of fungal species in environmental or clinical samples, being suitable for microorganisms which are difficult or even impossible to culture. OBJECTIVE In this study, we aimed to standardise an in situ hybridisation technique for the differentiation between the pathogenic species Paracoccidioides brasiliensis and Paracoccidioides lutzii, by using species-specific DNA probes targeting the internal transcribed spacer-1 (ITS-1) of the rRNA gene. METHODS Yeast and mycelial phase of each Paracoccidioides species, were tested by two different detection/differentiation techniques: TSA-FISH for P. brasiliensis with HRP (Horseradish Peroxidase) linked to the probe 5’ end; and FISH for P. lutzii with the fluorophore TEXAS RED-X® also linked to the probe 5’ end. After testing different protocols, the optimised procedure for both techniques was accomplished without cross-positivity with other pathogenic fungi. FINDINGS The in silico and in vitro tests show no reaction with controls, like Candida and Cryptococcus (in silico) and Histoplasma capsulatum and Aspergillus spp. (in vitro). For both phases (mycelial and yeast) the in situ hybridisation showed dots of hybridisation, with no cross-reaction between them, with a lower signal for Texas Red probe than HRP-TSA probe. The dots of hybridisation was confirmed with genetic material marked with 4’,6-diamidino-2-phenylindole (DAPI), visualised in a different filter (WU) on fluorescent microscopic. MAIN CONCLUSION Our results indicated that TSA-FISH and/or FISH are suitable for in situ detection and differentiation of Paracoccidioides species. This approach has the potential for future application in clinical samples for the improvement of paracoccidioidomycosis patients prognosis. PMID:28177048
Villar, L A; Restrepo, A
Paracoccidioides brasiliensis (ATCC 60885) produces conidia that, when plated in enriched media and incubated at 21-25 degrees C, give rise to a yeast-form variant (YRT) (ATCC 46678). The virulence of this variant for BALB/c mice has been compared with that of the yeast and conidia produced by the parent isolate. Adult female mice were inoculated intravenously with 10(7) viable cells of YRT or yeast, or 5 x 10(5) viable conidia. Mice were weighed weekly, and three mice were sacrificed at 1, 2, 3, 4 and 8 weeks post-inoculation and their organs weighed and prepared for determination of viable counts (colony forming units; c.f.u.). Animals infected with yeast cells or with conidia exhibited little weight loss in comparison with that seen in the YRT-infected mice which began to lose weight 1 week post-challenge. Organ weights increased in the YRT group, while the changes in the remaining two groups were minor. The c.f.u. from these organs also increased in the YRT-infected mice, whereas the infection in the yeast-and conidia-infected mice was self-limiting. A proportion (16.6%) of the YRT-infected animals died during the observation period. Conversely, no deaths occurred in the remaining groups. These results indicate that the YRT variant possesses increased virulence.
Background The pathogenic fungus Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis (PCM). This is a pulmonary mycosis acquired by inhalation of fungal airborne propagules that can disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside the host, P. brasiliensis may use the glyoxylate cycle for intracellular survival. Results Here, we provide evidence that the malate synthase of P. brasiliensis (PbMLS) is located on the fungal cell surface, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody was obtained against this protein. By using Confocal Laser Scanning Microscopy, PbMLS was detected in the cytoplasm and in the cell wall of the mother, but mainly of budding cells of the P. brasiliensis yeast phase. PbMLSr and its respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis with in vitro cultured epithelial cells A549. Conclusion These observations indicated that cell wall-associated PbMLS could be mediating the binding of fungal cells to the host, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection, behaving as an anchorless adhesin. PMID:20034376
Tamayo, Diana; Hernández, Orville; Muñoz-Cadavid, Cesar; Cano, Luz Elena; González, Angel
The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.
Brummer, E; Hanson, L H; Restrepo, A; Stevens, D A
The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis. Images PMID:2744848
da Fonseca, C A; Jesuino, R S; Felipe, M S; Cunha, D A; Brito, W A; Soares, C M
Paracoccidioides brasiliensis is a fungal pathogen of humans. To identify antigens from P. brasiliensis we fractionated a crude preparation of proteins from the fungus and detected the IgG reactive proteins by immunoblot assays of yeast cellular extracts with sera of patients with paracoccidioidomycosis (PCM). We identified and characterized six new antigens by amino acid sequencing and homology search analyses with other proteins deposited in a database. The newly characterized antigens were highly homologous to catalase, fructose-1,6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and triosephosphate isomerase from several sources. The characterized antigens presented preferential synthesis in yeast cells, the host fungus phase.
Rodríguez-Brito, Sabrina; Niño-Vega, Gustavo; San-Blas, Gioconda
Five Paracoccidioides brasiliensis isolates were grown in the presence of caspofungin (0 to 1 μg/ml). Inhibition of the yeast phase ranged from 20 to 65%, while in the mycelial form it ranged from 75% to 82%. Such variability was loosely related to the amount of cell wall β-1,3-glucan. No association with point mutations in the β-1,3-glucan synthase was detected. Caspofungin induced physical changes and cytoplasmic deterioration in both fungal phases. PMID:20937789
Valim, Clarissa X. R.; Basso, Luiz Roberto; dos Reis Almeida, Fausto B.; Reis, Thaila Fernanda; Damásio, André Ricardo Lima; Arruda, Luisa Karla; Martinez, Roberto; Roque-Barreira, Maria Cristina; Oliver, Constance; Jamur, Maria Célia; Coelho, Paulo Sergio Rodrigues
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection. PMID:23024763
Silva, Mirelle Garcia; Schrank, Augusto; Bailão, Elisa Flávia L.C.; Bailão, Alexandre Melo; Borges, Clayton Luiz; Staats, Charley Christian; Parente, Juliana Alves; Pereira, Maristela; Salem-Izacc, Silvia Maria; Mendes-Giannini, Maria José Soares; Oliveira, Rosely Maria Zancopé; Silva, Lívia Kmetzsch Rosa e; Nosanchuk, Joshua D.; Vainstein, Marilene Henning; de Almeida Soares, Célia Maria
Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways. PMID:21833306
Rocha, Antonio A; Malavazi, Iran; Goldman, Gustavo H; Puccia, Rosana
We show indirect evidences for the possible involvement of NIT2-like binding motifs in transcription modulation of the PbGP43 gene, which codes for an important antigen from the human fungal pathogen Paracoccidioides brasiliensis. This investigation was motivated by the finding of 23 NIT2-like sites within the proximal -2047 nucleotides of the PbGP43 5' intergenic region from the Pb339 isolate. They compose four clusters, two of them identical. We found four NIT2-containing probes that were positive in electrophoretic mobility shift assays and further analyzed them. PbGP43 could be modulated by nitrogen primary sources in Pb339, Pb3 and Pb18 isolates, as observed by reverse transcription (RT) real time-PCR. Gene reporter assays conducted in Aspergillus nidulans suggested that the minimal fragment responsible for nitrogen modulation lies within -480 bp of the PbGP43 gene. This is the first report on PbGP43 transcription modulation in response to nitrogen primary sources, which might help understand its regulation during infection.
Valim, Clarissa Xavier Resende; da Silva, Elaine Zayas Marcelino; Assis, Mariana Aprigio; Fernandes, Fabricio Freitas; Coelho, Paulo Sergio Rodrigues; Oliver, Constance; Jamur, Maria Célia
Background The fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients. Methodology/Principal Findings Endogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1. Conclusions/Significance The results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for
Restrepo, A; Salazar, M E; Cano, L E; Stover, E P; Feldman, D; Stevens, D A
Evidence that disease due to the thermally dimorphic fungus Paracoccidioides brasiliensis occurs post-puberty predominantly in males led us to hypothesize that hormonal factors critically affect its pathogenesis. We show here that estrogens inhibit mycelial- to yeast-form transformation of P. brasiliensis in vitro. Transformation of three isolates was inhibited to 71, 33, and 19% of the control values in the presence of 10(-10), 10(-8), and 10(-6) M 17 beta-estradiol, respectively. The synthetic estrogen diethylstilbestrol was active but less potent than estradiol, whereas testosterone, 17 alpha-estradiol, tamoxifen, and corticosterone were inactive. This function was specifically inhibited, since yeast-to-mycelium transformation, yeast growth, and yeast reproduction by budding were unaffected by 17 beta-estradiol. Of note is the fact that mycelium-to-yeast transformation occurs as the first step in vivo in the establishment of infection. The cytosol of the three isolates studied possesses a steroid-binding protein which has high affinity for 17 beta-estradiol. We believe that this binding protein represents a P. brasiliensis hormone receptor which can also recognize mammalian estrogens. We hypothesize that the ability of estrogen to decrease or delay mycelium-to-yeast transformation at the initial site of infection contributes to or is responsible for the marked resistance of females, and that the binder described is the molecular site of action. Images PMID:6500694
Derengowski, Lorena S; De-Souza-Silva, Calliandra; Braz, Shélida V; Mello-De-Sousa, Thiago M; Báo, Sônia N; Kyaw, Cynthia M; Silva-Pereira, Ildinete
Background Farnesol is a sesquiterpene alcohol produced by many organisms, and also found in several essential oils. Its role as a quorum sensing molecule and as a virulence factor of Candida albicans has been well described. Studies revealed that farnesol affect the growth of a number of bacteria and fungi, pointing to a potential role as an antimicrobial agent. Methods Growth assays of Paracoccidioides brasiliensis cells incubated in the presence of different concentrations of farnesol were performed by measuring the optical density of the cultures. The viability of fungal cells was determined by MTT assay and by counting the colony forming units, after each farnesol treatment. The effects of farnesol on P. brasiliensis dimorphism were also evaluated by optical microscopy. The ultrastructural morphology of farnesol-treated P. brasiliensis yeast cells was evaluated by transmission and scanning electron microscopy. Results In this study, the effects of farnesol on Paracoccidioides brasiliensis growth and dimorphism were described. Concentrations of this isoprenoid ranging from 25 to 300 μM strongly inhibited P. brasiliensis growth. We have estimated that the MIC of farnesol for P. brasiliensis is 25 μM, while the MLC is around 30 μM. When employing levels which don't compromise cell viability (5 to 15 μM), it was shown that farnesol also affected the morphogenesis of this fungus. We observed about 60% of inhibition in hyphal development following P. brasiliensis yeast cells treatment with 15 μM of farnesol for 48 h. At these farnesol concentrations we also observed a significant hyphal shortening. Electron microscopy experiments showed that, despite of a remaining intact cell wall, P. brasiliensis cells treated with farnesol concentrations above 25 μM exhibited a fully cytoplasmic degeneration. Conclusion Our data indicate that farnesol acts as a potent antimicrobial agent against P. brasiliensis. The fungicide activity of farnesol against this pathogen is
Albuquerque, Priscila C; Cordero, Radames J B; Fonseca, Fernanda L; Peres da Silva, Roberta; Ramos, Caroline L; Miranda, Kildare R; Casadevall, Arturo; Puccia, Rosana; Nosanchuk, Joshua D; Nimrichter, Leonardo; Guimaraes, Allan J; Rodrigues, Marcio L
The cell wall of the yeast form of the dimorphic fungus Paracoccidioides brasiliensis is enriched with α1,3-glucans. In Cryptococcus neoformans, α1,3-glucans interact with glucuronoxylomannan (GXM), a heteropolysaccharide that is essential for fungal virulence. In this study, we investigated the occurrence of P. brasiliensis glycans sharing properties with cryptococcal GXM. Protein database searches in P. brasiliensis revealed the presence of sequences homologous to those coding for enzymes involved in the synthesis of GXM and capsular architecture in C. neoformans. In addition, monoclonal antibodies (mAbs) raised to cryptococcal GXM bound to P. brasiliensis cells. Using protocols that were previously established for extraction and analysis of C. neoformans GXM, we recovered a P. brasiliensis glycan fraction composed of mannose and galactose, in addition to small amounts of glucose, xylose and rhamnose. In comparison with the C. neoformans GXM, the P. brasiliensis glycan fraction components had smaller molecular dimensions. The P. brasiliensis components, nevertheless, reacted with different GXM-binding mAbs. Extracellular vesicle fractions of P. brasiliensis also reacted with a GXM-binding mAb, suggesting that the polysaccharide-like molecule is exported to the extracellular space in secretory vesicles. An acapsular mutant of C. neoformans incorporated molecules from the P. brasiliensis extract onto the cell wall, resulting in the formation of surface networks that resembled the cryptococcal capsule. Coating the C. neoformans acapsular mutant with the P. brasiliensis glycan fraction resulted in protection against phagocytosis by murine macrophages. These results suggest that P. brasiliensis and C. neoformans share metabolic pathways required for the synthesis of similar polysaccharides and that P. brasiliensis yeast cell walls have molecules that mimic certain aspects of C. neoformans GXM. These findings are important because they provide additional evidence for
Almeida, Fausto; Antoniêto, Amanda Cristina Campos; Pessoni, André Moreira; Monteiro, Valdirene Neves; Alegre-Maller, Ana Claudia Paiva; Pigosso, Laurine Lacerda; Pereira, Maristela; Soares, Célia Maria de Almeida; Roque-Barreira, Maria Cristina
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America. It is caused by the temperature-dependent dimorphic fungus Paracoccidioides brasiliensis. The P. brasiliensis cell wall is a dynamic outer structure, composed of a network of glycoproteins and polysaccharides, such as chitin, glucan and N-glycosylated proteins. These glycoproteins can interact with the host to affect infection rates, and are known to perform other functions. We inhibited N-linked glycosylation using tunicamycin (TM), and then evaluated the expression of P. brasiliensis genes related to cell wall remodeling. Our results suggest that cell wall synthesis related genes, such as β-1,3-glucanosyltransferase (PbGEL3), 1,3-β-D-glucan synthase (PbFKS1), and α-1,4-amylase (PbAMY), as well as cell wall degrading related genes, such as N-acetyl-β-D-glucosaminidase (PbNAG1), α-1,3-glucanase (PbAGN), and β-1,3-glucanase (PbBGN1 and PbBGN2), have their expression increased by the N-glycosylation inhibition, as detected by qRT-PCR. The observed increases in gene expression levels reveal possible compensatory mechanisms for diminished enzyme activity due to the lack of glycosylation caused by TM. PMID:27226767
Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N
Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.
Goldani, L Z; Maia, A L; Sugar, A M
We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates.
Goldani, L Z; Maia, A L; Sugar, A M
We cloned and sequenced a species-specific 110-bp DNA fragment from Paracoccidioides brasiliensis. The DNA fragment was generated by PCR with primers complementary to the rat beta-actin gene under a low annealing temperature. Comparison of the nucleotide sequence, after excluding the primers, with those in the GenBank database identified approximately 60% homology with an exon of a major surface glycoprotein gene from Pneumocystis carinii and a fragment of unknown function in Saccharomyces cerevisiae chromosome VIII. By Southern hybridization analysis, the 32P-labelled fragment detected 1.0- and 1.9-kb restriction fragments within whole-cell genomic DNA of P. brasiliensis digested with HindIII and PstI, respectively, but failed to hybridize to genomic DNAs from Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Aspergillus fumigatus, Saccharomyces cerevisiae, Pneumocystis carinii, rat tissue, or humans under low-stringency hybridization conditions. Additionally, the specific DNA fragment from three different P. brasiliensis isolates (Pb18, RP18, RP17) was amplified by PCR with primers mostly complementary to nonactin sequences of the 110-bp DNA fragment. In contrast, there were no amplified products from other fungus genomic DNAs previously tested, including Histoplasma capsulatum. To date, this is the first species-specific DNA fragment cloned from P. brasiliensis which might be useful as a diagnostic marker for the identification and classification of different P. brasiliensis isolates. PMID:7650207
Taborda, Carlos P.; da Silva, Marcelo B.; Nosanchuk, Joshua D.; Travassos, Luiz R.
Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (“ghosts”) can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs. PMID:18777637
Jimenez-Finkel, B E; Murphy, J W
In naturally acquired paracoccidioidomycosis, patients have depressed in vivo and in vitro cell-mediated immune (CMI) responses to Paracoccidioides brasiliensis antigen. In addition, it has been reported that these patients have significant levels of circulating paracoccidioidal antigen in their sera. The primary purpose of this investigation was to assess the effects of P. brasiliensis antigen on the CMI responses in a mouse model. On the basis of findings with other fungal agents, we predicted that circulating paracoccidioidal antigen may be inducing suppressor cells which modulate the CMI response. In this study, we show (i) that a soluble P. brasiliensis culture filtrate antigen (Pb.Ag) emulsified in complete Freund adjuvant and injected subcutaneously into mice induces reasonably high levels of delayed-type hypersensitivity (DTH) in CBA/J mice; (ii) that Pb.Ag elicits DTH reactions specific for P. brasiliensis when injected into footpads of immunized mice; and (iii) that an intravenous injection of Pb.Ag induces a population of lymph node and spleen cells which, upon adoptive transfer, suppress the afferent limb of the DTH response to paracoccidioidal antigen. The afferent suppressor cells can be detected in spleens as early as 5 days after Pb.Ag treatment, are present in significant numbers by 7 days in both spleens and lymph nodes, and are virtually absent by 14 days. In contrast, at 14 days after antigen injection, efferent suppressor cells were detected in spleens and lymph nodes. The Pb.Ag-induced afferent suppressor cells specifically inhibit the antiparacoccidioidal DTH response. They are nylon wool-nonadherent cells, and their activity is abrogated by anti-Thy-1 and complement treatment, indicating that they are T lymphocytes. The phenotype of these afferent suppressor T cells is L3T4+ Lyt-1+2- I-J+. The Pb.Ag-specific suppressor cells described in this paper are similar to the Ts1 cells in the azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, and
RESTREPO, Angela; CANO, Luz Elena; GONZALEZ, Ángel
SUMMARY Research on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensis interactions. PMID:26465363
Morais, Elis Araujo; Martins, Estefânia Mara do Nascimento; Boelone, Jankerle Neves; Gomes, Dawidson Assis; Goes, Alfredo Miranda
Paracoccidioidomycosis (PCM) is a systemic mycosis in which the host response to the infectious agent typically consists of a chronic granulomatous inflammatory process. This condition causes lesions that impair lung function and lead to chronic pulmonary insufficiency resulting from fibrosis development, which is a sequel and disabling feature of the disease. The rPb27 protein has been studied for prophylactic and therapeutic treatment against PCM. Previous studies from our laboratory have shown a protective effect of rPb27 against PCM. However, these studies have not determined whether rPb27 immunization prevents lung fibrosis. We therefore conducted this study to investigate fibrosis resulting from infection by Paracoccidioides brasiliensis in the lungs of animals immunized with rPb27. Animals were immunized with rPb27 and subsequently infected with a virulent strain of P. brasiliensis. Fungal load was evaluated by counting colony-forming units, and Masson's trichrome staining was performed to evaluate fibrosis at 30 and 90 days post-infection. The levels of CCR7, active caspase 3, collagen and cytokines were analyzed. At the two time intervals mentioned, the rPb27 group showed lower levels of fibrosis on histology and reduced levels of collagen and the chemokine receptor CCR7 in the lungs. CCR7 was detected at higher levels in the control groups that developed very high levels of pulmonary fibrosis. Additionally, the immunized groups showed high levels of active caspase 3, IFN-γ, TGF-β and IL-10 in the early phase of P. brasiliensis infection. Immunization with Pb27, in addition to its protective effect, was shown to prevent pulmonary fibrosis.
Fernandes, Larissa; Araújo, Marcus A M; Amaral, André; Reis, Viviane Castelo Branco; Martins, Natália F; Felipe, M S
The human fungal pathogen Paracoccidioides brasiliensis is an ascomycete that displays a temperature-dependent dimorphic transition, appearing as a mycelium at 22 degrees C and as a yeast at 37 degrees C, this latter being the virulent form. We report on the in silico search made of the P. brasiliensis transcriptome-expressed sequence tag database for components of signaling pathways previously known to be involved in morphogenesis and virulence in other species of fungi, including Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Using this approach, it was possible to identify several protein cascades in P. brasiliensis, such as i) mitogen-activated protein kinase signaling for cell integrity, cell wall construction, pheromone/mating, and osmo-regulation, ii) the cAMP/PKA system, which regulates fungal development and virulence, iii) the Ras protein, which allows cross-talking between cascades, iv) calcium-calmodulin-calcineurin, which controls cell survival under oxidative stress, high temperature, and membrane/cell wall perturbation, and v) the target of rapamycin pathway, controlling cell growth and proliferation. The ways in which P. brasiliensis responds to the environment and modulates the expression of genes required for its survival and virulence can be inferred through comparison with other fungi for which this type of data is already available.
Terçarioli, Gisela Ramos; Bagagli, Eduardo; Reis, Gabriela Martins; Theodoro, Raquel Cordeiro; Bosco, Sandra De Moraes Gimenes; Macoris, Severino Assis da Graça; Richini-Pereira, Virgínia Bodelão
Background Paracoccidioides brasiliensis ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions. Methods Soils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. P. brasiliensis was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region. Results The soil texture does not affect fungus development and the growth is more abundant on/in soil saturated with water. Some soil samples inhibited the development of P. brasiliensis, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil. Conclusion P. brasiliensis may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al. PMID:17953742
Bianchini, A.A.C.; Petroni, T.F.; Fedatto, P.F.; Bianchini, R.R.; Venancio, E.J.; Itano, E.N.; Ono, M.A.
The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis. PMID:24031350
Marcos, Caroline Maria; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; Assato, Patrícia Akemi; Singulani, Junya de Lacorte; Lopez, Angela Maria; Tamayo, Diana Patricia; Hernandez-Ruiz, Orville; McEwen, Juan G; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa
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. PMID:26646480
Singer-Vermes, L M; Burger, E; Calich, V L; Modesto-Xavier, L H; Sakamoto, T N; Sugizaki, M F; Meira, D A; Mendes, R P
The pathogenicity and immunogenicity of six recently isolated Paracoccidioides brasiliensis samples derived from patients presenting distinct and well defined clinical forms of paracoccidioidomycosis (PCM) were compared as to their virulence, tropism to different organs and ability to induce specific cellular and humoral immune response in susceptible (B10.A) inbred mice. Isolates Pb44 and Pb47 were obtained from acute cases, Pb50 from a chronic severe form, Pb45 from a chronic moderate case and both Pb56 and Pb57 from chronic mild forms of PCM. Pathogenicity and tropism of each fungal sample were evaluated by LD50% estimation, examination of gross lesions on various organs at 2, 4, 12 and 16 weeks post-infection, and by colony-forming unit (CFU) counts in the lungs at week 16 post-infection of mice. Fungal tropism in human PCM and in B10.A mice was always dissociated. A well defined relationship between virulence of the fungal sample and the clinical findings of the correspondent patient was not evident, although a tendency to higher LD50% and less intense paracoccidioidic lesions was observed in mice infected with Pb56 and Pb57. The specific DTH response patterns varied according to the infectant sample, but positive DTH reactions at the beginning of the infection and a tendency to anergy or low DTH responses at week 12 and/or week 16 post-infection were always observed. A correspondence between the DTH response in humans and in mice was noticeable only when the isolates from the most benign cases (Pb56 and Pb57) were considered. The specific antibody patterns in mice and in the correspondent patients were also not analogous. Collectively, these results indicate that an association between the fungal pathogenicity and immunogenicity in the human disease and in susceptible mice was discernible only when isolates obtained from very mild cases (Pb56 and Pb57) were considered. PMID:8033408
Ferreira, Isabelle; Ferreira-Strixino, Juliana; Castilho, Maiara L.; Campos, Claudia B. L.; Tellez, Claudio; Raniero, Leandro
Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25 °C in vitro) and as yeast cells in the human host (or at 37 °C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach's for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-β-glucan usually found in mycelia and 1,3-α-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions.
Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A
A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.
Silva, Marcelo Barbosa da; Thomaz, Luciana; Marques, Alexandre Ferreira; Svidzinski, Artur E; Nosanchuk, Josh D; Casadevall, Arturo; Travassos, Luiz R; Taborda, Carlos P
Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.
Arango, R; Restrepo, A
The mycelial and yeast forms of the fungus Paracoccidioides brasiliensis were cultured in a chemically defined liquid medium with different iron concentrations and the growth measured spectrophotometrically. The iron binding capacity of culture supernatants was measured by a colorimetric assay. Both the mycelial and yeast forms were able to grow in media containing trace amounts of iron (0.02 mgl-1) but when the iron chelant 1.10 phenantroline was added there was a delay in the initiation of growth of the yeast and almost total inhibition of the mycelium. When iron excess was added to media containing phenantroline, this inhibitory effect was reversed, partially for the mycelial, and completely for the yeast form. For both mycelial and yeast forms, the iron binding capacity of the culture supernatants was greater in media with low iron concentrations.
San Blas, F; Centeno, S
N-methyl-N'-nitro-N-nitrosoguanidine, which is known to be a very effective mutagen in many systems, was used to induce mutants in the yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9, an imperfect fungus. Forty-three auxotrophic and 27 prototrophic morphological mutants were isolated after treatment with 50 mug of nitrosoguanidine per ml in 0.1 M citrate buffer, pH 5.0. Auxotrophic mutants required primarily either amino acids, purines, or pyrimidines. Some auxotrophs were also morphological mutants. The main morphological difference from the parental strain was the texture or the color of the yeast-like colonies. Only one prototrophic morphological mutant differed in the size and form of the yeastlike cells when compared with the parental strain. Suxotrophic mutants were used in pairwise combination to attempt heterokaryon formation without success. Images PMID:830638
Martínez, J. R. Ramírez
Details of the sequential morphological changes occurring during yeastlike to mycelial-form conversion of the dimorphic pathogen Paracoccidioides brasiliensis are described and illustrated by photomicrographs. Conversion of yeastlike to hyphal morphology was initiated by changing the temperature of incubation from 37 to 23 C. Production by the parent yeastlike cells of elongated buds developing into hyphae started to be conspicuous after 24 hr of incubation at 23 C. After 120 hr of incubation, growth was almost exclusively filamentous. Direct transformation of parent yeastlike cells into hyphae was not observed. Dry weight increased continuously during the conversion process in spite of the gradual disappearance of the parent yeastlike cells. Concurrent studies showed that changes in ribonucleic acid and deoxyribonucleic acid content per unit dry weight are about the same whether the yeastlike cells are undergoing conversion at 23 C or growing normally at 37 C, and that deoxyribonucleic acid synthesis is apparently required for bud formation in both cases. Images PMID:5541529
Ramírez Martínez, J R
Details of the sequential morphological changes occurring during yeastlike to mycelial-form conversion of the dimorphic pathogen Paracoccidioides brasiliensis are described and illustrated by photomicrographs. Conversion of yeastlike to hyphal morphology was initiated by changing the temperature of incubation from 37 to 23 C. Production by the parent yeastlike cells of elongated buds developing into hyphae started to be conspicuous after 24 hr of incubation at 23 C. After 120 hr of incubation, growth was almost exclusively filamentous. Direct transformation of parent yeastlike cells into hyphae was not observed. Dry weight increased continuously during the conversion process in spite of the gradual disappearance of the parent yeastlike cells. Concurrent studies showed that changes in ribonucleic acid and deoxyribonucleic acid content per unit dry weight are about the same whether the yeastlike cells are undergoing conversion at 23 C or growing normally at 37 C, and that deoxyribonucleic acid synthesis is apparently required for bud formation in both cases.
Vallejo, Milene C; Matsuo, Alisson L; Ganiko, Luciane; Medeiros, Lia C Soares; Miranda, Kildare; Silva, Luiz S; Freymüller-Haapalainen, Edna; Sinigaglia-Coimbra, Rita; Almeida, Igor C; Puccia, Rosana
Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 × g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying α-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.
Kanetsuna, Fuminori; Carbonell, Luis M.
Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315–1320. 1966.—Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form. PMID:5924267
Kanetsuna, F; Carbonell, L M
Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315-1320. 1966.-Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form.
Carbonell, L M; Kanetsuna, F; Gil, F
Short and thick fibers were observed on the outer surface of the yeast phase of Paracoccidioides brasiliensis, and long and thin fibers were seen on the inner surface. The long fibers disappear with chitinase treatment and are composed of chitin. The short fibers disappear under alkali treatment and are composed of alpha-glucan. Comparisons with alpha-(1 --> 3)-glucan isolated from Aspergillus niger and Polyporus betulinus and with chitin from fungal origin support our point of view.
Mattar-Filho, R; Azevedo, M O; Pereira, M; Jesuino, R S; Salem-Izacc, S M; Brito, W A; Gesztesi, J L; Soares, R B; Felipe, M S; Soares, C M
Expression of the 43 kDa glycoprotein (gp43) was analysed in several Paracoccidioides brasiliensis isolates. Using one- and two-dimensional analysis of crude cellular extracts, it was shown that protein expression in yeast and mycelium was dependent on the isolate analysed. In two strains, in both yeast and mycelium cells. gp43 was present, whereas expression was restricted to the yeast phase of two other strains. The clinical implications of this phase-specific gp43 expression are uncertain.
Pereira, M; Felipe, M S; Brígido, M M; Soares, C M; Azevedo, M O
1,3-beta-D-glucan is a fungal cell wall polymer synthesized by the multi-subunit enzyme 1,3-beta-D-glucan synthase. A subunit of this integral membrane protein was first described as the product of the FKS1 gene from Saccharomyces cerevisiae using echinocandin mutants. Other FKS1 genes were also reported for Candida albicans, Aspergillus nidulans and Cryptococcus neoformans. Here, we report the nucleotide sequence of the first homologous FKS gene cloned from the pathogenic fungus Paracoccidioides brasiliensis. An open reading frame of 5942 bp was identified in the complete sequence, interrupted by two putative introns, the first close to the 5' end and the second close to the 3' end of the gene. A promoter region is also described containing consensus sequences such as canonical TATA and CAAT boxes and, possibly, multiple sites for glucose regulation by creA protein. The deduced sequence of 1926 amino acid show more than 85% similarity to FksAp from A. nidulans, and 71% to Fks1p and Fks2p from S. cerevisiae. Computational analysis of P. brasiliensis Fks1p suggests a similar structure to transmembrane proteins, such as FksAp, with the presence of two domains composed by hydrophobic helices that limit the putative highly hydrophilic catalytic domain within the cytoplasm.
Calcagno, Ana María; Niño-Vega, Gustavo; San-Blas, Felipe; San-Blas, Gioconda
Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of P. brasiliensis. PMID:9620409
Salazar, M E; Restrepo, A; Stevens, D A
Conidia produced by Paracoccidioides brasiliensis are inhibited by mammalian estrogens in their in vitro conversion into yeast-form cells. This was demonstrated with four different isolates. In these experiments, conversion was reduced to 10.7 and 34.4% of the control values by 17-beta-estradiol at 10(-6) and 10(-8) M, respectively. At the same concentrations, the synthetic estrogen diethylstilbestrol was slightly less inhibitory. In contrast, other sex hormones and analogs, i.e., testosterone, 17-alpha-estradiol, tamoxifen, and hydroxytamoxifen, had no effect on conidium-to-yeast conversion. Previous studies have shown that estrogens similarly inhibit mycelium-to-yeast-form transition in P. brasiliensis. Conidia, and not mycelial fragments, are believed to be the natural infectious propagules. These findings with conidia support the hypothesis that estrogens, affecting the initial host-parasite interactions by suppressing conversion to the parasitic form of the organism, are, at least in part, responsible for the greater resistance of females to paracoccidioidomycosis.
Salazar, M E; Restrepo, A; Stevens, D A
Conidia produced by Paracoccidioides brasiliensis are inhibited by mammalian estrogens in their in vitro conversion into yeast-form cells. This was demonstrated with four different isolates. In these experiments, conversion was reduced to 10.7 and 34.4% of the control values by 17-beta-estradiol at 10(-6) and 10(-8) M, respectively. At the same concentrations, the synthetic estrogen diethylstilbestrol was slightly less inhibitory. In contrast, other sex hormones and analogs, i.e., testosterone, 17-alpha-estradiol, tamoxifen, and hydroxytamoxifen, had no effect on conidium-to-yeast conversion. Previous studies have shown that estrogens similarly inhibit mycelium-to-yeast-form transition in P. brasiliensis. Conidia, and not mycelial fragments, are believed to be the natural infectious propagules. These findings with conidia support the hypothesis that estrogens, affecting the initial host-parasite interactions by suppressing conversion to the parasitic form of the organism, are, at least in part, responsible for the greater resistance of females to paracoccidioidomycosis. Images PMID:3343055
Longo, Larissa V. G.; Ganiko, Luciane; Lopes, Felipe G.; Matsuo, Alisson L.; Almeida, Igor C.; Puccia, Rosana
Background Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. Methodology/Principal Findings Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. Conclusions/Significance The extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions. PMID:22745761
Costa, Alessandra A; Gómez, Francisco J; Pereira, Maristela; Felipe, M Sueli S; Jesuino, Rosália S A; Deepe, George S; de Almeida Soares, Célia M
We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.
Felipe, Maria Sueli S; Andrade, Rosângela V; Arraes, Fabrício B M; Nicola, André M; Maranhão, Andréa Q; Torres, Fernando A G; Silva-Pereira, Ildinete; Poças-Fonseca, Márcio J; Campos, Elida G; Moraes, Lídia M P; Andrade, Patrícia A; Tavares, Aldo H F P; Silva, Simoneide S; Kyaw, Cynthia M; Souza, Diorge P; Pereira, Maristela; Jesuíno, Rosália S A; Andrade, Edmar V; Parente, Juliana A; Oliveira, Gisele S; Barbosa, Mônica S; Martins, Natália F; Fachin, Ana L; Cardoso, Renato S; Passos, Geraldo A S; Almeida, Nalvo F; Walter, Maria Emília M T; Soares, Célia M A; Carvalho, Maria José A; Brígido, Marcelo M
Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.
Jesuino, Rosália S A; Azevedo, Maristela O; Felipe, M Sueli S; Pereira, Maristela; De Almeida Soares, Célia M
We report the cloning and sequence analysis of a genomic clone encoding a Paracoccidioides brasiliensis ClpB chaperone homologue (PbClpB). The clpb gene was identified in a lambda Dash II library. Sequencing of Pbclpb revealed a long open reading frame capable of encoding a 792 amino acid, 87.9 kDa protein, pI of 5.34. The predicted polypeptide contains several consensus motifs of the ClpB proteins. Canonical sequences such as two putative nucleotide-binding sites, chaperonins ClpA/B signatures and highly conserved casein kinase phosphorylation domains are present. ClpB is 69% to 49% identical to members of the ClpB family from several organisms from prokaryotes to eukaryotes. The transcript of PbclpB was detected as a mRNA species of 3.0 kb, preferentially expressed in the yeast parasitic phase of the fungus. A 89 kDa protein was also detected in yeast cells of P. brasiliensis.
Chagas, Ronney Fernandes; Bailão, Alexandre Melo; Fernandes, Kátia Flávia; Winters, Michael S; Pereira, Maristela; Soares, Célia Maria de Almeida
Catalases are essential components of the cellular equipment to cope with oxidative stress. Here we have purified a highly abundant catalase P of Paracoccidioides brasiliensis (PbCatP) that is preferentially expressed in the parasitic yeast phase. This oxidative stress-induced protein was isolated from yeast cells grown in the presence of 15 mM of hydrogen peroxide (H(2)O(2)). We have used consecutive steps of protein precipitation and gel filtration chromatography to achieve the purified protein. Protein purification was validated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and bioinformatics analysis. The purified enzyme showed strong similarity to small-subunit catalases. Like most monofunctional catalases, PbCatP is a homotetramer, resistant to inactivation by acidic conditions, temperature and denaturants. Furthermore, the kinetic behaviour of catalase P was observed to be different at low compared to high H(2)O(2) concentrations. The results demonstrated that a purified PbCatP is a homotetrameric enzyme, classified as a small subunit catalase.
Cruz, Aline H da Silva; Brock, Matthias; Zambuzzi-Carvalho, Patrícia F; Santos-Silva, Ludier K; Troian, Rogério F; Góes, Alfredo M; Soares, Célia M de Almeida; Pereira, Maristela
The glyoxylate cycle plays an essential role for anaplerosis of oxaloacetate during growth of microorganisms on carbon sources such as acetate or fatty acids and has been shown to contribute to virulence of several pathogens. Here, we investigated the transcriptional and post-translational regulation of the glyoxylate cycle key enzyme isocitrate lyase (PbICL) in the human pathogenic fungus Paracoccidioides brasiliensis. Although sequence analyses on fungal isocitrate lyases revealed a high phylogenetic conservation, their regulation seems to differ significantly. Closely related Aspergillus species regulate the glyoxylate cycle at the transcriptional level, whereas Pbicl was constitutively expressed in yeast cells. However, only low PbICL activity was detected when cells were grown in the presence of glucose. Two-dimensional gel analyses with subsequent antibody hybridization revealed constitutive production of PbICL, but low PbICL activity on glucose coincided with extensive protein phosphorylation. Since an in vitro dephosphorylation of PbICL from glucose grown cells strongly increased ICL activity and resembled the phosphorylation pattern of highly active acetate grown cells, post-translational modification seems the main mechanism regulating PbICL activity in yeast cells. In agreement, a transfer of yeast cells from glucose to acetate medium increased PbICL activity without requirement of de novo protein synthesis. Thus, inactivation of PbICL by phosphorylation is reversible, denoting a new strategy for the rapid adaptation to changing environmental conditions.
CARBONELL, L M; RODRIGUEZ, J
Carbonell, Luis M. (Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela), and Joaquín Rodríguez. Transformation of mycelial and yeast forms of Paracoccidioides brasiliensis in cultures and in experimental inoculations. J. Bacteriol. 90:504-510. 1965.-Experimental transformations of mycelial to yeast and yeast to mycelial forms in culture, and mycelial to yeast forms in tissue, were studied. All the transitional forms that appeared in culture were also seen in tissue, but in fewer number. Most of the hyphae in culture were transformed into yeast, but only a few in tissue. Yeast appeared in testicle around the 3rd day after inoculation, but on the 10th day in subcutaneous tissue. Pathogenicity of mycelium was high, since yeast was found in almost all of the organs inoculated with mycelium. Histologically, an acute inflammation occurred first, owing to the inoculation of mycelium, followed by a giant-cell granuloma with abundant hyphae detritus. These giant cells almost disappeared about 10 days after inoculation, giving place to a second giant-cell granuloma with yeast forms.
Carbonell, Luis M.; Rodríguez, Joaquín
Carbonell, Luis M. (Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela), and Joaquín Rodríguez. Transformation of mycelial and yeast forms of Paracoccidioides brasiliensis in cultures and in experimental inoculations. J. Bacteriol. 90:504–510. 1965.—Experimental transformations of mycelial to yeast and yeast to mycelial forms in culture, and mycelial to yeast forms in tissue, were studied. All the transitional forms that appeared in culture were also seen in tissue, but in fewer number. Most of the hyphae in culture were transformed into yeast, but only a few in tissue. Yeast appeared in testicle around the 3rd day after inoculation, but on the 10th day in subcutaneous tissue. Pathogenicity of mycelium was high, since yeast was found in almost all of the organs inoculated with mycelium. Histologically, an acute inflammation occurred first, owing to the inoculation of mycelium, followed by a giant-cell granuloma with abundant hyphae detritus. These giant cells almost disappeared about 10 days after inoculation, giving place to a second giant-cell granuloma with yeast forms. Images PMID:14329466
[Production and standardization of Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus antigens to be used in immunodiagnosis. Comparison between immunodiffusion and immunoelectrophoresis].
Ferreira-da-Cruz, M F; Galvão-Castro, B; Wanke, B
Soluble antigens (Ag) from Paracoccidioides brasiliensis, Histoplasma capsulatum and Aspergillus fumigatus were prepared and standardized by double immunodiffusion (DID) and immunoelectroosmophoresis (IEOP). No difference in sensitivity was observed between the two techniques; 100% of standard patient sera were positive with P. brasiliensis and A. fumigatus Ag and 83.3% were positive with H. capsulatum Ag. The specificity of the tests was verified testing 96 sera from patients with paracoccidioidomycosis, histoplasmosis, systemic candidiasis, sporotrichosis, tuberculosis, lung cancer, visceral or cutaneous leishmaniasis and 18 sera from healthy individuals. All the three antigens were 100% specific with the DID (using the identification pattern indicated by the confluence of test serum with standard serum precipitin lines as a positive criterium). However in the IEOP, the specificity varied with each Ag. Positive reactions with P. brasiliensis Ag were observed in 16.7% of histoplasmosis sera and in 10% of cutaneous leishmaniasis sera. On the other hand 31.8% of paracoccidioidomycosis and 10% of cutaneous leishmaniasis sera reacted with H. capsulatum Ag. The high sensitivity and specificity of the DID test, its easy reproducibility and low cost, led us to consider it highly appropriate as a routine procedure for the screening of patients with respiratory infections.
Marcos, Caroline Maria; de Oliveira, Haroldo Cesar; da Silva, Julhiany de Fátima; Assato, Patricia Akemi; Yamazaki, Daniella Sayuri; da Silva, Rosângela Aparecida Moraes; Santos, Cláudia Tavares; Santos-Filho, Norival Alves; Portuondo, Deivys Leandro; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa
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.
Venancio, Emerson J; Daher, Bruno S; Andrade, Rosângela V; Soares, Célia M A; Pereira, Ildinete Silva; Felipe, Maria Sueli S
Kexin-like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin-like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin-like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin-like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5' non-coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE-like regulating genes involved in thermo-dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3' non-coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin-like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin-like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin-like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the
Díez, Soraya; Gómez, Beatriz L; Restrepo, Angela; Hay, Rod J; Hamilton, Andrew J
The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.
Peron, G; Fernandes, F F; Landgraf, T N; Martinez, R; Panunto-Castelo, A
Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.
Ortiz, B L; Díez, S; Urán, M E; Rivas, J M; Romero, M; Caicedo, V; Restrepo, A; McEwen, J G
Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5alpha, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 microgram/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic
Ortiz, B. L.; Díez, S.; Urán, M. E.; Rivas, J. M.; Romero, M.; Caicedo, V.; Restrepo, A.; McEwen, J. G.
Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5α, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 μg/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic
Caro, Erika; Gonzalez, Angel; Muñoz, César; Urán, Marta E; Restrepo, Angela; John Hamilton, Andrew; Elena Cano, Luz
This study addresses the recognition of laminin by Paracoccidioides brasiliensis conidia, as well as its possible role in the adherence of conidia to A549 cells. Adherence of conidia to immobilized laminin was shown to be specific, as anti-laminin antibodies, soluble laminin or the laminin-derived peptides IKVAV and CDPGYIGSR inhibited this interaction. RGD containing peptides and various monosaccharides had no effect on adherence, with the exception of N-acetylneuraminic acid. Pre-treatment of conidia with fibrinogen and fibronectin, but not with BSA, also resulted in significant inhibition, suggesting that P. brasiliensis conidia might cross-recognize host proteins involved in colonization. In assays using transmission electron microscopy, we observed internalization of conidia 30 min after exposition to A549 cells. Laminin present on the surface of A549 cells shown to serve as mediator of this interaction, with a significant decrease in fungal adherence when the epithelial cells were pre-treated with anti-laminin antibodies or when conidia were pre-incubated with either soluble laminin or the laminin-specific peptides. Together these results suggest that the recognition of laminin by P. brasiliensis conidia is a key process in the interaction with pulmonary epithelial cells, where this extracellular matrix protein acts as bridging molecule.
Background The aim of this study was to isolate and identify the antifungal compounds from the extracts of Schinus terebinthifolius (Anacardiaceae) against clinical isolates of the pathogenic fungus Paracoccidioides brasiliensis. Methods The hexane and dichlomethane fractions from leaves and stems of S. terebinthifolius were fractionated using several chromatography techniques to afford four compounds. Results The compounds isolated from S. terebinthifolius were identified as schinol (1), a new biphenyl compound, namely, 4'-ethyl-4-methyl-2,2',6,6'-tetrahydroxy[1,1'-biphenyl]-4,4'-dicarboxylate (2), quercetin (3), and kaempferol (4). Compounds 1 and 2 were active against different strains of P. brasiliensis, showing a minimal inhibitory concentration value against the isolate Pb B339 of 15.6 μg/ml. The isolate Pb 1578 was more sensitive to compound 1 with a MIC value of 7.5 μg/ml. Schinol presented synergistic effect only when combined with itraconazole. The compounds isolated from S. terebinthifolius were not able to inhibit cell wall synthesis or assembly using the sorbitol assay. Conclusion This work reveals for the first time the occurrence of compound 2 and discloses activity of compounds 1 and 2 against several clinical isolates of P. brasiliensis. These results justify further studies to clarify the mechanisms of action of these compounds. PMID:20939907
Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing--Paracoccidioides cerebriformis revisited.
Cavalcanti, Sarah Desirée Barbosa; Levi, José Eduardo; Dantas, Kátia Cristina; Martins, José Eduardo Costa
Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.
Morais, Flavia V.; Barros, Tânia F.; Fukada, Márcio K.; Cisalpino, Patrícia S.; Puccia, Rosana
The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine. PMID:11060052
Barbosa, Mônica S; Cunha Passos, Daniela A; Felipe, M Sueli S; Jesuíno, Rosália S A; Pereira, Maristela; de Almeida Soares, Célia M
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays important roles in various cellular processes. Here we report the sequence and analysis of a novel developmentally regulated gene and cDNA (Pbgadph), encoding a GAPDH homologue (PbGAPDH), of the pathogenic dimorphic fungus Paracoccidioides brasiliensis. We have analyzed the protein, the cDNA and genomic sequences to provide insights into the structure, function, and potential regulation of PbGAPDH. That Pbgapdh encodes PbGAPDH was demonstrated by micro-sequencing of the native protein homologue isolated from the fungus proteome. The deduced amino acid sequence of Pbgapdh showed identity to those of from other species (88-76%). Phylogenetic analysis indicated that GAPDH could be useful for the determination of evolutionary relationships. Expression of the Pbgapdh gene and the cognate protein were developmentally regulated in phases of P. brasiliensis, with a higher expression in the yeast parasitic phase and was induced during the transition from mycelium to yeast and decreased during the reverse process, transition from yeast to mycelium.
Tamayo, Diana; Muñoz, Jose F; Torres, Isaura; Almeida, Agostinho J; Restrepo, Angela; McEwen, Juan G; Hernández, Orville
HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.
Cadavid, D.; Restrepo, A.
The natural habitat of Paracoccidioides brasiliensis, the aetiologic agent of paracoccidioidomycosis, has not been determined. Consequently, the events leading to the acquisition of infection remain controversial. To identify factors associated with infection in endemic areas we conducted a survey in three rural communities in Colombia where we had previously diagnosed paracoccidioidomycosis in children. Permanent residents were surveyed taking into consideration environmental and occupational variables. Skin tests were used to classify subjects as infected or non-infected. Variables found associated with infection were: (i) community A: previous residence around Porce river and agriculture in vegetable gardens; (ii) community C: frequent use of specific water sources; (iii) community V: housekeeping activities, and (iv) total group: age > 25 years and contact with bats. Residents in communities with higher prevalence of infection were older, had more complex residence history, and referred more contact with armadillos than residents of communities with lower infection. PMID:8348926
Cadavid, D; Restrepo, A
The natural habitat of Paracoccidioides brasiliensis, the aetiologic agent of paracoccidioidomycosis, has not been determined. Consequently, the events leading to the acquisition of infection remain controversial. To identify factors associated with infection in endemic areas we conducted a survey in three rural communities in Colombia where we had previously diagnosed paracoccidioidomycosis in children. Permanent residents were surveyed taking into consideration environmental and occupational variables. Skin tests were used to classify subjects as infected or non-infected. Variables found associated with infection were: (i) community A: previous residence around Porce river and agriculture in vegetable gardens; (ii) community C: frequent use of specific water sources; (iii) community V: housekeeping activities, and (iv) total group: age > 25 years and contact with bats. Residents in communities with higher prevalence of infection were older, had more complex residence history, and referred more contact with armadillos than residents of communities with lower infection.
Flavia Popi, Ana Flavia; Lopes, José Daniel; Mariano, Mario
Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues. The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell-microorganism interactions. Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known. Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb. Phagocytosis of Pb, live or heat-killed, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages. Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts.
Imai, T; Sano, A; Mikami, Y; Watanabe, K; Aoki, F H; Branchini, M L; Negroni, R; Nishimura, K; Miyaji, M
Internal transcribed spacer (ITS) genes including the 5.8S ribosomal (r)RNA of Paracoccidioides brasiliensis were amplified and the DNA sequences were determined. Based on a comparison of the sequence information, a new polymerase chain reaction (PCR) primer pair was designed for specific amplification of DNA for P. brasiliensis. This primer pair amplified a 418-bp DNA sequence and was 100% successful in identifying 29 strains of P. brasiliensis (including the reference strains) isolated from the regions of Brazil, Costa Rica, Japan, Argentina or from different sources. The results of specificity tests of these primers to compare the fungus with those of Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei are also reported.
Villar, L A; Salazar, M E; Restrepo, A
Incubation of Paracoccidioides brasiliensis conidia at 20-25 degrees C (RT) results in two types of colonies, mycelial (M) and yeast (YRT). A study of the latter colonies was undertaken. Conidia were plated in complex (BHI) and chemically-defined media (CDCM), with and without fetal calf serum (FCS). Incubation was carried out at 21 degrees C for 4 weeks. The mean number of YRT colonies was approximately 18.5% on BHI agar. Selected YRT colonies were transferred to liquid CDCM, incubated 7 days at 21 degrees C and transferred to solid media; YRT appearance was preserved in 95% of the colonies if media were FCS-supplemented; otherwise, most colonies reverted to the M form. When FCS was replaced by bovine albumin or by alpha-globulin, 63% and 68% respectively of the colonies obtained after plating YRT cells, became mycelial. Comparative morphologic studies of both YRT and yeasts grown at 37 degrees C suggested that there were no major differences between these two types of yeast cells when size and budding were taken into consideration. The results indicate that in this particular variant, dimorphism is not exclusively temperature-dependent.
Puccia, R; Travassos, L R
Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL) were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 125I-labeled antigens. In IPP, PCM sera and other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and 29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrate epitopes in these reactions, we compared the reactivities of sera from several patients with native and deglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. The results suggest that greater than 85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrate epitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were only weakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could be significantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrate epitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate than when it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivity with PCM sera and support the existence of conformational peptide epitopes. Images PMID:1722220
Jesuino, R S; Soares, R de B; Salem-Izacc, S M; Pereira, M; Felipe, M S; Soares, C M
The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.
Izacc, S M; Gomez, F J; Jesuino, R S; Fonseca, C A; Felipe, M S; Deepe, G S; Soares, C M
A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).
Mendes, Graziele; Gonçalves, Vívian N; Souza-Fagundes, Elaine M; Kohlhoff, Markus; Rosa, Carlos A; Zani, Carlos L; Cota, Betania B; Rosa, Luiz H; Johann, Susana
Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values ≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.
Mendes, Graziele; Gonçalves, Vívian N; Souza-Fagundes, Elaine M; Kohlhoff, Markus; Rosa, Carlos A; Zani, Carlos L; Cota, Betania B; Rosa, Luiz H; Johann, Susana
Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity. PMID:27008375
Fernandes, Alison M.; Sluzevich, Jason C.
Pulmonary nocardiosis is a severe and uncommon opportunistic infection caused by Nocardia species. We present a patient with cryptogenic organizing pneumonia who was receiving long-term immunosuppressive therapy, whose treatment course was complicated by cutaneous and pulmonary nocardiosis. Tissue cultures confirmed Nocardia brasiliensis. Nocardiosis should be a diagnostic consideration for patients treated with long-term immunosuppression who have worsening pulmonary symptoms and relapsing pustular skin lesions. PMID:28348912
de Sá, Nívea P; Cisalpino, Patrícia S; Tavares, Luciana C; Espíndola, Leandro; Borelli, Beatriz M; Barbeira, Paulo Js; Cardoso Perdigão, Gabriele de Matos; Souza-Fagundes, Elaine M; Rosa, Carlos A; Pizzolatti, Moacir G; Johann, Susana
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, yet few therapeutic options exist. Our aim was to search for new compounds with high efficacy, low toxicity, shorter treatment time and affordable cost. We studied two synthetic 6-quinolinyl chalcones, 3b and 3e, to determine their effects on VERO cells, antifungal activity, survival curve, interaction with other drugs and phenotypic effects against several isolates of Paracoccidioides spp. In this study, we verified that the compounds were not toxic, exhibited superior in vitro activity compared with that shown by trimethoprim-sulfamethoxazole, and after 5 days of treatment, decreased the fungal cell viability by approximately 70%. Additionally, no interactions were observed between the tested compounds and other drugs. We also found that these compounds induced morphological changes, such as shriveling of cells, fragmentation of the plasma membrane and cytoplasmic disorganization in vitro. The changes observed by microscopy assays corroborate the observation made with propidium iodide, where the number of cells stained with the compounds was higher than that observed after amphotericin B treatment. We observed an increase in the efflux of K(+) and a loss of intracellular contents in cells treated with 3b and 3e, confirming their effects on fungal membranes. However, damage to the membrane was not associated with a decrease in membrane ergosterol levels. The experimental evidences showed no direct indications of cellular wall damage caused by these compounds. Thus, these results confirm the antifungal potential of 3b and 3e against Paracoccidioides spp. with possible action on the membrane.
Baltazar, Ludmila Matos; Werneck, Silvia Maria Cordeiro; Soares, Betânia Maria; Ferreira, Marcus Vinicius L.; Souza, Danielle G.; Pinotti, Marcos; Santos, Daniel Assis
Paracoccidioidomycosis (PCM) is a public health concern in Latin America and South America that when not correctly treated can lead to patient death. In this study, the influence of melanin produced by Paracoccidioides spp. on the effects of treatment with antimicrobial photodynamic inhibition (aPI) and antifungal drugs was evaluated. aPI was performed using toluidine blue (TBO) as a photosensitizer and a 630-nm light-emitting diode (LED) light. The antifungals tested were itraconazole and amphotericin B. We evaluated the effects of each approach, aPI or antifungals, against nonmelanized and melanized yeast cells by performing susceptibility tests and by quantifying oxidative and nitrosative bursts during the experiments. aPI reduced nonmelanized cells by 3.0 log units and melanized cells by 1.3 log units. The results showed that melanization protects the fungal cell, probably by acting as a scavenger of nitric oxide and reactive oxygen species, but not of peroxynitrite. Melanin also increased the MICs of itraconazole and amphotericin B, and the drugs were fungicidal for nonmelanized and fungistatic for melanized yeast cells. Our study shows that melanin production by Paracoccidioides yeast cells serves a protective function during aPI and treatment with itraconazole and amphotericin B. The results suggest that melanin binds to the drugs, changing their antifungal activities, and also acts as a scavenger of reactive oxygen species and nitric oxide, but not of peroxynitrite, indicating that peroxynitrite is the main radical that is responsible for fungal death after aPI. PMID:25896704
Immunohistochemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoccidioides brasiliensis in biopsy material and partial characterization by using species-specific monoclonal antibodies.
Figueroa, J I; Hamilton, A; Allen, M; Hay, R
Two murine monoclonal antibodies (MAbs) specific to Paracoccidioides brasiliensis (as determined by enzyme-linked immunosorbent assay [ELISA] and Western blot [immunoblot]) were produced by using a modification of standard hybridization protocols, with cyclophosphamide included as an immunomodulator to abolish responses to highly cross-reactive immunodominant epitopes. MAbs PS14 and PS15 are two different clones which exhibit similar characteristics by ELISA and Western blot. They are directed against a 22- to 25-kDa antigen which is present in P. brasiliensis and which could not be identified in other dimorphic fungi by ELISA or Western blot. Partial purification of the antigen was accomplished by isoelectric focusing, and deglycosylation studies suggested that the 22- to 25-kDa antigen is a glycoprotein with a pI of between 4.5 and 5 and that O-linked sugars may be part of the recognized epitope. The MAbs stained the cytoplasm of P. brasiliensis yeast and hyphal cells in cryostat sections of fresh cultures of the fungus. In addition, the MAbs stained the wall of paracoccidioidomycotic granulomas, as well as the cytoplasm of the fungus, as determined by the use of immunofluorescence, immunoperoxidase, and immuno-alkaline phosphatase staining techniques in paraffin-embedded sections of human biopsy material, and they failed to stain granulomas resulting from other clinical conditions. These findings suggest that these MAbs have potential use in the immunohistochemical identification of P. brasiliensis. Images PMID:8077405
Cruz, R. C.; Werneck, S. M. C.; Oliveira, C. S.; Santos, P. C.; Soares, B. M.; Santos, D. A.
MIC assays with Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, had been conducted with variable protocols, employing both macrodilution and microdilution tests and including differences in inoculum preparation, media used, incubation periods, and temperatures. Twenty-one clinical and environmental isolates of Paracoccidioides were tested using amphotericin B, itraconazole, ketoconazole, fluconazole, sulfamethoxazole, sulfamethoxazole-trimethoprim, and terbinafine, according to the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards, document M27-A2, 2002), with modifications such as three medium formulations (RPMI 1640 medium, McVeigh and Morton [MVM] medium, and modified Mueller-Hinton [MMH] medium), two incubation temperatures (room temperature [25 to 28°C] and 37°C), and three incubation periods (7, 10, and 15 days). The antifungal activities were also classified as fungicidal or fungistatic. The best results were obtained after 15 days of incubation, which was chosen as the standard incubation time. The MICs for most individual isolates grown for the same length of time at the same temperature varied with the different media used (P < 0.05). Of the isolates, 81% showed transition from the yeast to the mycelial form in RPMI 1640 medium at 37°C, independent of the presence of antifungals. MMH medium appears to be a suitable medium for susceptibility testing of antifungal drugs with P. brasiliensis, except for sulfamethoxazole and the combination of sulfamethoxazole-trimethoprim, for which the MVM medium yielded better results. The incubation temperature influenced the MICs, with, in general, higher MICs at 25°C (mycelial form) than at 37°C (P < 0.05). Based on our results, we tentatively propose a microdilution assay protocol for susceptibility testing of antifungal drugs against Paracoccidioides. PMID:23175254
Villalobos-Duno, Héctor; San-Blas, Gioconda; Paulinkevicius, Maryan; Sánchez-Martín, Yolanda; Nino-Vega, Gustavo
α-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade α-1,3-glucan and allow remodeling of the cell wall, α-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal α-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (α-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall α-1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl α-1,3-glucan (SCMG, chemically modified from P. brasiliensis α-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1Δ strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at
Souza, A R; Gesztesi, J -L; Moraes, J Z; Cruz, C R B; Sato, J; Mariano, M; Lopes, J D
Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network. PMID:9764601
Reinoso, Cristina; Sorais, Françoise; Niño-Vega, Gustavo A; Fermiñán, Encarnación; San-Blas, Gioconda; Domínguez, Angel
A genomic clone encoding the Paracoccidioides brasiliensis orotidine monophosphate decarboxylase gene (PbrURA3) was isolated by screening a subgenomic plasmid DNA library of this fungus, using a PCR amplification product of the gene as a probe. Sequence analysis revealed that the gene contains an open reading frame of 855 bp with a single intron (162 bp), and encodes a putative 285 amino acids polypeptide of estimated molecular weight 31.1 kDa and isoelectric point 6.5. The deduced amino acid sequence predicted a 73.4% identity with orotidine monophosphate decarboxylase of Aspergillus nidulans. Functionality of the gene was demonstrated by transformation into a Saccharomyces cerevisiae ura3 null mutant.
Bernardi, Thais; da Silva, Julhiany de Fátima; Vicentin, Juliana; de Oliveira, Haroldo Cesar; Assato, Patricia Akemi; Marcos, Caroline Maria; de Paula E Silva, Ana Carolina Alves; da Silva, Rosangela Aparecida Moraes; Regasini, Luis Octávio; Silva, Dulce Helena Siqueira; da Silva Bolzani, Vanderlan; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares
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.
Silva, M R; de Paiva e Rosália, L F; Jesuino, S A
Male albino mice were inoculated intravenously with 0.5 x 10(7) viable yeast forms of P. brasiliensis (strain 2052). These animals were treated with two doses of ketoconazole (50 and 100 mg/kg) during fifty days and the sacrificed. We studied the presence of P. brasiliensis, the inflammatory granulomatous response of liver and spleen and the anti P. brasiliensis delayed hypersensitivity response measured by the footpad test after 48 hours. It was observed that: 1. animals infected and treated with ketoconazole showed reduction in the number of fungi in the organs studied; 2. there was no difference in the number of granulomas among animals treated and non-treated; 3. the cutaneous delayed tests intensity was similar in all animals.
Santos, Mônica O; Pereira, Maristela; Felipe, Maria Sueli S; Jesuino, Rosalia Santos A; Ulhoa, Cirano J; Soares, Renata de Bastos A; Soares, Celia Maria de A
A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.
Salinas-Carmona, M C
Nocardia brasiliensis is a Gram-positive bacterium that lives as a saprophyte in soil. In this article the physical properties, chemical composition and taxonomic position of this species is reviewed. Human infections and an experimental model of actinomycetoma in BALB/c mice as well as the host-immune response is described.
Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.
Toledo, M S; Levery, S B; Straus, A H; Suzuki, E; Momany, M; Glushka, J; Moulton, J M; Takahashi, H K
Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These
Cano, L E; Gomez, B; Brummer, E; Restrepo, A; Stevens, D A
Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells.
Cano, L E; Gomez, B; Brummer, E; Restrepo, A; Stevens, D A
Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells. PMID:8132359
Arantes, Thales Domingos; Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Bosco, Sandra de Moraes Gimenes; Bagagli, Eduardo
Background Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiological agents of Paracoccidioidomycosis (PCM), and are easily isolated from human patients. However, due to human migration and a long latency period, clinical isolates do not reflect the spatial distribution of these pathogens. Molecular detection of P. brasiliensis and P. lutzii from soil, as well as their isolation from wild animals such as armadillos, are important for monitoring their environmental and geographical distribution. This study aimed to detect and, for the first time, evaluate the genetic diversity of P. brasiliensis and P. lutzii for Paracoccidioidomycosis in endemic and non-endemic areas of the environment, by using Nested PCR and in situ hybridization techniques. Methods/Principal Findings Aerosol (n = 16) and soil (n = 34) samples from armadillo burrows, as well as armadillos (n = 7) were collected in endemic and non-endemic areas of PCM in the Southeastern, Midwestern and Northern regions of Brazil. Both P. brasiliensis and P. lutzii were detected in soil (67.5%) and aerosols (81%) by PCR of Internal Transcribed Spacer (ITS) region (60%), and also by in situ hybridization (83%). Fungal isolation from armadillo tissues was not possible. Sequences from both species of P. brasiliensis and P. lutzii were detected in all regions. In addition, we identified genetic Paracoccidioides variants in soil and aerosol samples which have never been reported before in clinical or armadillo samples, suggesting greater genetic variability in the environment than in vertebrate hosts. Conclusions/Significance Data may reflect the actual occurrence of Paracoccidioides species in their saprobic habitat, despite their absence/non-detection in seven armadillos evaluated in regions with high prevalence of PCM infection by P. lutzii. These results may indicate a possible ecological difference between P. brasiliensis and P. lutzii concerning their wild hosts. PMID:27045486
de Paula e Silva, A. C. A.; Oliveira, H. C.; Silva, J. F.; Sangalli-Leite, F.; Scorzoni, L.; Fusco-Almeida, A. M.
CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs. PMID:23345296
García-Hernández, M; Castro-Corona, M A; Segoviano-Ramírez, J C; Brattig, N W; Medina-De la Garza, C E
We tested whether diethylcarbamazine (DEC) or ivermectin (IVM), both antiparasitic drugs with reported immunomodulatory properties, were able to affect the immune system to potentiate host defense mechanisms and protect against actinomycetoma in a mouse model. Male BALB/c mice of 10-12 weeks of age were injected with either Nocardia brasiliensis or saline solution. Recorded were the effects of a treatment by DEC (6 mg/kg per os daily for one week) or IVM (200 μg/kg subcutaneously on days 1 and 3) on (i) the development of mycetoma lesion, (ii) the expression of reactive oxygen intermediates (ROI) by phagocytes, (iii) the proliferation index of lymphocytes and (iv) antibody production of IgG and IgM. After an initial lesion in all mice, DEC inhibited a full development and progression of actinomycetoma resulting in a reduced lesion size (p < 0.001). IVM had no inhibitory effect on the development of mycetoma. Furthermore, DEC treatment was associated with a significant enhancement of ROI expression (p < 0.05) by polymorphonuclear neutrophils at day 3 after infection. Lymphocyte proliferation in response to N. brasiliensis antigens and concanavalin A in DEC-treated group was higher than in non-treated group at day 21 and 28 postinfection (p < 0.01). Significant changes in antibody response were not observed. By all parameters tested, DEC was superior to IVM regarding immunostimulatory potency. In conclusion, DEC expressed an in vivo influence on the immune status during the infection by N. brasiliensis leading to retrogression of the mycetoma and increasing cellular immune responses. Our findings may indicate a potential use of DEC as a putative adjuvant in infectious disease or vaccination.
Dos Reis Almeida, Fausto Bruno; Pigosso, Laurine Lacerda; de Lima Damásio, André Ricardo; Monteiro, Valdirene Neves; de Almeida Soares, Célia Maria; Silva, Roberto Nascimento; Roque-Barreira, Maria Cristina
The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-β-d-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and β-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or β-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and β-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells. © 2013 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:24155051
Dos Reis Almeida, Fausto Bruno; Pigosso, Laurine Lacerda; de Lima Damásio, André Ricardo; Monteiro, Valdirene Neves; de Almeida Soares, Célia Maria; Silva, Roberto Nascimento; Roque-Barreira, Maria Cristina
The cell wall of Paracoccidioides brasiliensis, which consists of a network of polysaccharides and glycoproteins, is essential for fungal pathogenesis. We have previously reported that N-glycosylation of proteins such as N-acetyl-β-D-glucosaminidase is required for the growth and morphogenesis of P. brasiliensis. In the present study, we investigated the influence of tunycamicin (TM)-mediated inhibition of N-linked glycosylation on α- and β-(1,3)-glucanases and on α-(1,4)-amylase in P. brasiliensis yeast and mycelium cells. The addition of 15 µg/ml TM to the fungal cultures did not interfere with either α- or β-(1,3)-glucanase production and secretion. Moreover, incubation with TM did not alter α- and β-(1,3)-glucanase activity in yeast and mycelium cell extracts. In contrast, α-(1,4)-amylase activity was significantly reduced in underglycosylated yeast and mycelium extracts after exposure to TM. In spite of its importance for fungal growth and morphogenesis, N-glycosylation was not required for glucanase activities. This is surprising because these activities are directed to wall components that are crucial for fungal morphogenesis. On the other hand, N-glycans were essential for α-(1,4)-amylase activity involved in the production of malto-oligosaccharides that act as primer molecules for the biosynthesis of α-(1,3)-glucan. Our results suggest that reduced fungal α-(1,4)-amylase activity affects cell wall composition and may account for the impaired growth of underglycosylated yeast and mycelium cells.
The PbMDJ1 gene belongs to a conserved MDJ1/LON locus in thermodimorphic pathogenic fungi and encodes a heat shock protein that localizes to both the mitochondria and cell wall of Paracoccidioides brasiliensis.
Batista, Wagner L; Matsuo, Alisson L; Ganiko, Luciane; Barros, Tânia F; Veiga, Thiago R; Freymüller, Edna; Puccia, Rosana
J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline beta-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5' region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.
Morimoto, M; Yamada, M; Arizono, N; Hayashi, T
The effects of lactic dehydrogenase virus (LDV) infection on the protective immune responses to the nematode Nippostrongylus brasiliensis were studied. Mice with chronic LDV infection showed significantly higher levels of parasite egg production than non-LDV-infected (control) mice after N. brasiliensis infection. Concurrent LDV infection also suppressed peripheral blood eosinophilia and the lung mastocytosis induced by this nematode. LDV infection showed higher expression levels of the interferon-gamma (IFN-gamma) mRNA in lymph nodes compared with control mice before N. brasiliensis infection. In addition, the IgG2a production in LDV-infected mice was higher than that in control mice before and after N. brasiliensis infection. These results suggest that LDV infection modulates protective immune responses against N. brasiliensis infection by the activation of T-helper type 1 cells. PMID:9659227
Hoving, Jennifer C.; Nieuwenhuizen, Natalie; McSorley, Henry J.; Ndlovu, Hlumani; Bobat, Saeeda; Kimberg, Matti; Kirstein, Frank; Cutler, Anthony J.; DeWals, Benjamin; Cunningham, Adam F.; Brombacher, Frank
In this study, B cell function in protective TH2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4+ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4+ T cell-mediated protective immunity against N. brasiliensis infection. PMID:24204255
Jalali, Hossnieh Kafshdar; Salamatzadeh, Abdolreza; Jalali, Arezou Kafshdar; Kashani, Hamed Haddad; Asbchin, Salman Ahmadi; Issazadeh, Khosro
The main drawback of current antibiotic therapies is the emergence and rapid increase in antibiotic resistance. Nocardiae are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Nocardia brasiliensis was reported as antibiotic producer. The purpose of the study was to determine antibacterial activity of N. brasiliensis PTCC 1422 against isolated Enterobacteriaceae from urinary tract infections (UTIs). The common bacteria from UTIs were isolated from hospital samples. Antimicrobial susceptibility test was performed for the isolated pathogens using Kirby-Bauer disk diffusion method according to clinical and Laboratory Standards Institute guideline. Antagonistic activity of N. brasiliensis PTCC 1422 was examined with well diffusion methods. Supernatant of N. brasiliensis PTCC 1422 by submerged culture was analyzed with gas chromatography-mass spectrometry. Isolated strains included Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Proteus mirabilis. The most common pathogen isolated was E. coli (72.5%). Bacterial isolates revealed the presence of high levels of antimicrobial resistances to ceftriaxone and low levels of resistance to cephalexin. Supernatant of N. brasiliensis PTCC 1422 showed antibacterial activity against all of the isolated microorganisms in well diffusion method. The antibiotic resistance among the uropathogens is an evolving process, so a routine surveillance to monitor the etiologic agents of UTI and the resistance pattern should be carried out timely to choose the most effective empirical treatment by the physicians. Our present investigation indicates that the substances present in the N. brasiliensis PTCC 1422 could be used to inhibit the growth of human pathogen. Antibacterial resistance among bacterial uropathogen is an evolving process. Therefore, in the field on the need of re-evaluation of empirical treatment of UTIs, our present. The study has demonstrated that N. brasiliensis PTCC 1422 has a high potential
Theodoro, Raquel Cordeiro; Teixeira, Marcus de Melo; Felipe, Maria Sueli Soares; Paduan, Karina dos Santos; Ribolla, Paulo Martins; San-Blas, Gioconda; Bagagli, Eduardo
Background Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. Methodology/Principal Findings Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11–32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. Conclusions/Significance The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides. PMID:22666382
IL-13 has a prominent role in host defense against the gastrointestinal nematode, Nippostrongylus brasiliensis; however, the role of IL-13 alpha2 in the immune and functional response to enteric infection is not known. In the current study, we investigated changes in smooth muscle and epithelial ce...
Salinas-Carmona, Mario C.; Pérez-Rivera, Isabel
An experimental model of infection with Nocardia brasiliensis, used as an example of a facultative intracellular pathogen, was tested. N. brasiliensis was injected into the rear foot pads of BALB/c mice to establish an infection. Within 30 days, infected animals developed a chronic actinomycetoma infection. Batch cultures of N. brasiliensis were used to purify P61, P38, and P24 antigens; P61 is a catalase, and P38 is a protease with strong caseinolytic activity. Active and passive immunizations of BALB/c mice with these three purified soluble antigens were studied. Protection was demonstrated for actively immunized mice. However, immunity lasted only 30 days. Other groups of immunized mice were bled at different times, and their sera were passively transferred to naive recipients that were then infected with N. brasiliensis. Sera collected 5, 6, and 7 days after donor immunization conferred complete, long-lasting protection. The protective effect of passive immunity decreased when sera were collected 2 weeks after donor immunization. However, neither the early sera (1-, 2-, and 3-day sera) nor the later sera (30- or 45-day sera) prevented the infection. Hyperimmune sera with the highest levels of immunoglobulin G (IgG) to N. brasiliensis antigens did not protect at all. The antigens tested induced two IgM peaks. The first peak was present 3 days after immunization but was not antigen specific and did not transfer protection. The second peak was evident 7 days after immunization, was an IgM response, was antigen specific, and conferred protection. This results clearly demonstrate that IgM antibodies protect the host against a facultative intracellular bacterium. PMID:15385456
Revol, Agnès; Espinoza-Ruiz, Marisol; Medina-Villanueva, Igor; Salinas-Carmona, Mario Cesar
Nocardia brasiliensis is the main agent of actinomycetoma in Mexico, but little is known about its virulence and molecular pathogenic pathways. These facultative intracellular bacteria are able to survive and divide within the host phagocytic cells, in part by neutralizing the reactive oxygen intermediates. Superoxide dismutase (SOD) participates in the intracellular survival of several bacterial species and, in particular, constitutes one of Nocardia asteroides virulence factors. To clarify SOD participation in the N. brasiliensis early infective process, we report its isolation and the consequent comparison of its transcript level. A 630 bp polymerase chain reaction fragment that included most of the coding sequence of N. brasiliensis sodA was cloned. A competitive assay was developed, allowing comparison of bacterial sod expression in exponential culture and 1 h after infecting peritoneal macrophages from BALB/c mice. At that time, there were viable bacteria in the macrophages. The intracellular bacteria presented a clear decrease in their sod transcript amount, although their 16S rRNA (used as an internal control) and hsp levels were maintained or slightly increased, respectively. These results indicate that sodA transcription is not maintained within the SOS bacterial response induced by phagosomal conditions. Further kinetics will be necessary to precisely define sod transcriptional regulation during N. brasiliensis intra-macrophage growth.
Rosas-Taraco, Adrian G.; Perez-Liñan, Amira R.; Bocanegra-Ibarias, Paola; Perez-Rivera, Luz I.
Nocardia brasiliensis is an intracellular microorganism and the most common etiologic agent of actinomycetoma in the Americas. Several intracellular pathogens induce an immunosuppressive microenvironment through increases in CD4+ Foxp3+ regulatory T cells (Treg), thus downregulating other T-cell subpopulations and assuring survival in the host. In this study, we determined whether N. brasiliensis modulates T-lymphocyte responses and their related cytokine profiles in a murine experimental model. We also examined the relationship between N. brasiliensis immunomodulation and pathogenesis and bacterial survival. In early infection, Th17/Tc17 cells were increased at day 3 (P < 0.05) in footpad tissue and spleen. Treg subpopulations peaked at days 7 and 15 (P < 0.01) in the footpad and spleen, respectively. Transforming growth factor β1 (TGF-β1) and interleuki-10 (IL-10) are cytokines known for their immunosuppressive effects. During early and chronic infections, these cytokines were elevated with increased TGF-β1 levels from days 3 to 30 (P < 0.01) and sustained IL-10 expression throughout infection compared to uninfected mice. IL-6 production was increased at day 3 (P < 0.01), whereas gamma interferon (IFN-γ), IL-17A, and IL-23 levels were highest at day 15 postinfection (P < 0.01) when a decrease in the bacterial load (>1 log) was also observed (P < 0.05). After these changes, at 30 to 60 days postinfection, IFN-γ production was decreased, whereas the expression of anti-inflammatory cytokines and the bacterial load again increased (P < 0.05). The increment in Treg cells and the related cytokine profile correlated with reduced inflammation at day 15 (P < 0.05) in the footpad. We conclude that N. brasiliensis modulates the immune system to induce an immunosuppressive microenvironment that benefits its survival during the chronic stage of infection. PMID:22547544
Oliveira, Tiago G; Carvalho-Costa, Filipe A; Gomes, Taís F; Sarquis, Otília; Sposina, Ricardo; Lima, Marli M
The aim of this work was to study the interaction between Trypanosoma cruzi-1 and Triatoma brasiliensis. A group of 1st instar nymphs was initially fed on T. cruzi-infected mice and a control group was fed on uninfected mice. From the second feeding onwards, both groups were otherwise fed on non-infected mice. The resulting adults were grouped in pairs: infected male/uninfected female, uninfected male/infected female, infected male and female and uninfected male/uninfected female. The infection affected only the 1st instar nymphs, which took significantly more time to reach the 2nd instar than uninfected nymphs. The differences in the molting time between the infected and uninfected nymphs from the 2nd to the 5th instars were not statistically significant. Both groups presented similar rates of nymphal mortality and reproductive performance was not significantly affected by infection in any of the treatments.
Tamayo, Diana; Muñoz, José F; Lopez, Ángela; Urán, Martha; Herrera, Juan; Borges, Clayton L; Restrepo, Ángela; Soares, Celia M; Taborda, Carlos P; Almeida, Agostinho J; McEwen, Juan G; Hernández, Orville
The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.
Tamayo, Diana; Muñoz, José F.; Lopez, Ángela; Urán, Martha; Herrera, Juan; Borges, Clayton L.; Restrepo, Ángela; Soares, Celia M.; Taborda, Carlos P.; Almeida, Agostinho J.; McEwen, Juan G.; Hernández, Orville
The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms. PMID:26963091
Ribeiro, S C; Ferreira, F S; Brito, S V; Teles, D A; Ávila, R W; Almeida, W O; Anjos, L A; Guarnieri, M C
The parameters of infection by lung parasites from two sympatric lizards, Mabuya arajara and Anolis brasiliensis, from the Atlantic Rainforest of the lower slope of Chapada do Araripe in Northeastern Brazil were analyzed between September, 2009 and July, 2010. A total of 202 lizards were collected. 125 specimens were from Mabuya arajara and 77 from Anolis brasiliensis. M. arajara was infected by the pentastomid Raillietiella mottae while A. brasiliensis was infected by the nematode Rhabdias sp., with an overall prevalence of 1.6% and 28.6%, respectively. The mean intensity of infection by Rhabdias sp. was 3.63 ± 2.58 (range 1-15). The body size and sex of lizards did not influence the intensity of infection by Rhabdias sp. The overall prevalence was also not different between males and females hosts in A. brasiliensis. Both Anolis brasiliensis and Mabuya arajara represent a new host to Rhabdias sp. and Raillietiella mottae, respectively.
de Oliveira, Haroldo C.; Assato, Patrícia A.; Marcos, Caroline M.; Scorzoni, Liliana; de Paula E Silva, Ana C. A.; Da Silva, Julhiany De Fátima; Singulani, Junya de Lacorte; Alarcon, Kaila M.; Fusco-Almeida, Ana M.; Mendes-Giannini, Maria J. S.
Paracoccidioides brasiliensis and P. lutzii are etiologic agents of paracoccidioidomycosis (PCM), an important endemic mycosis in Latin America. During its evolution, these fungi have developed characteristics and mechanisms that allow their growth in adverse conditions within their host through which they efficiently cause disease. This process is multi-factorial and involves host–pathogen interactions (adaptation, adhesion, and invasion), as well as fungal virulence and host immune response. In this review, we demonstrated the glycoproteins and polysaccharides network, which composes the cell wall of Paracoccidioides spp. These are important for the change of conidia or mycelial (26°C) to parasitic yeast (37°C). The morphological switch, a mechanism for the pathogen to adapt and thrive inside the host, is obligatory for the establishment of the infection and seems to be related to pathogenicity. For these fungi, one of the most important steps during the interaction with the host is the adhesion. Cell surface proteins called adhesins, responsible for the first contact with host cells, contribute to host colonization and invasion by mediating this process. These fungi also present the capacity to form biofilm and through which they may evade the host’s immune system. During infection, Paracoccidioides spp. can interact with different host cell types and has the ability to modulate the host’s adaptive and/or innate immune response. In addition, it participates and interferes in the coagulation system and phenomena like cytoskeletal rearrangement and apoptosis. In recent years, Paracoccidioides spp. have had their endemic areas expanding in correlation with the expansion of agriculture. In response, several studies were developed to understand the infection using in vitro and in vivo systems, including alternative non-mammal models. Moreover, new advances were made in treating these infections using both well-established and new antifungal agents. These
Alves, Fernanda Lourenço; Ribeiro, Mariceli Araújo; Hahn, Rosane Christine; de Melo Teixeira, Marcus; de Camargo, Zoilo Pires; Cisalpino, Patrícia Silva; Marini, Marjorie Mendes
Studies comparing Paracoccidioides brasiliensis and Paracoccidioides lutzii have shown that these fungi have significant genomic differences that may have implications in the clinical manifestation, diagnosis, and treatment of paracoccidioidomycosis caused by them. Thus, molecular typing methods are required that can distinguish between various species of Paracoccidioides. The aim of this study was to explore the potential use as molecular markers of the transposable elements Trem A-H recently identified and characterized in the genus Paracoccidioides as a means of differentiating the species. We take advantage of the abundance and distribution of these transposons in the Paracoccidioides genomes to develop a simple and highly reproducible polymerase chain reaction (PCR)-based technique. Furthermore we compare the performance of this test with two other molecular markers already in use to identify these fungi.
Salinas-Carmona, M C; Welsh, O; Casillas, S M
We previously identified three immunodominant antigens obtained from a Nocardia brasiliensis cell extract and recognized by sera from mycetoma patients (M. C. Salinas-Carmona, L. Vera, O. Welsh, and M. Rodríguez, Zentralbl. Bakteriol. 276:390-397, 1992). In the present work, we obtained a crude extract from a mass culture of N. brasiliensis HUJEG-1 and purified two immunodominant antigens, the 26- and 24-kDa proteins, by using simple physiochemical techniques. With these antigens, we developed a conventional solid-phase enzyme-linked immunosorbent assay and tested 30 serum samples from mycetoma patients, 29 from tuberculosis patients, 24 from a leprosy group, and 31 from healthy individuals. Our results show for the first time statistically significant differences in serology among these groups. All mycetoma patients with a positive culture for N. brasiliensis had absorbance values higher than 0.3. On the other hand, the mycobacterium-infected patients as well as the healthy individuals all had absorbance values below that level. Moreover, we found a close correlation between the clinical condition of the mycetoma patients and the anti-26- and anti-24-kDa protein antibody concentrations. We therefore propose the use of this assay in routine clinical laboratories to confirm the diagnosis of N. brasiliensis infection in human mycetoma cases. In addition, the possible application of this assay in the serodiagnosis of Nocardia asteroides infection is also discussed.
Salinas-Carmona, M C; Welsh, O; Casillas, S M
We previously identified three immunodominant antigens obtained from a Nocardia brasiliensis cell extract and recognized by sera from mycetoma patients (M. C. Salinas-Carmona, L. Vera, O. Welsh, and M. Rodríguez, Zentralbl. Bakteriol. 276:390-397, 1992). In the present work, we obtained a crude extract from a mass culture of N. brasiliensis HUJEG-1 and purified two immunodominant antigens, the 26- and 24-kDa proteins, by using simple physiochemical techniques. With these antigens, we developed a conventional solid-phase enzyme-linked immunosorbent assay and tested 30 serum samples from mycetoma patients, 29 from tuberculosis patients, 24 from a leprosy group, and 31 from healthy individuals. Our results show for the first time statistically significant differences in serology among these groups. All mycetoma patients with a positive culture for N. brasiliensis had absorbance values higher than 0.3. On the other hand, the mycobacterium-infected patients as well as the healthy individuals all had absorbance values below that level. Moreover, we found a close correlation between the clinical condition of the mycetoma patients and the anti-26- and anti-24-kDa protein antibody concentrations. We therefore propose the use of this assay in routine clinical laboratories to confirm the diagnosis of N. brasiliensis infection in human mycetoma cases. In addition, the possible application of this assay in the serodiagnosis of Nocardia asteroides infection is also discussed. Images PMID:8263174
Dias, Maria Fernanda R. G.; Filgueira, Absalom L.; de Souza, Wanderley
Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils' degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.
Salinas-Carmona, Mario C; de la Cruz-Galicia, Guadalupe; Pérez-Rivera, Isabel; Solís-Soto, Juan M; Segoviano-Ramirez, Juan C; Vázquez, Anna Velia; Garza, Mario A
Rheumatoid arthritis is an autoimmune disease that affects human beings worldwide. Infections have been associated to autoimmune diseases because their ability to induce a dominant cytokine response. Joint inflammation has been related to Th1 response because they induce high expression of proinflammatory cytokines TNF-alpha, IL-1, IFN-gamma. MRL/lpr mice spontaneously develop an autoimmune disease affecting joints, kidneys, etc. We compared incidence and severity of arthritis, antibody response, cytokine production, in mice infected with bacteria or helminthes in the Murphy Roths Large (MRL)lpr mice. Infections with helminthes Heligmosomoides polygyrus, Nippostrongylus brasiliensis or bacteria Nocardia brasiliensis and Staphylococcus aureus were studied. IL-4, IFN-gamma and IgG1, IgG2a antibody productions were determined. IFN-gamma was increased in all groups, the highest production was observed after bacterial infection; IL-4 production was higher after helminthes infection. IgG1 sera levels were increased in the helminthes infected group. IgG2a sera concentration was stimulated by bacterial infection. The histopathology showed that 100% of bacterial infected mice developed arthritis and severe tissue damage such as cartilage erosion and bone destruction. Animals infected with parasites showed a decreased incidence and severity of arthritis. Severity of tissue damage in joints is correlated with increased numbers of lymphocytes and macrophages immunoreactive to proinflammatory cytokines.
Sarquis, Otilia; Carvalho-Costa, Filipe A; Oliveira, Lívia Silva; Duarte, Rosemere; D Andrea, Paulo Sergio; de Oliveira, Tiago Guedes; Lima, Marli Maria
We assessed some ecological parameters of Triatoma brasiliensis in rock piles in the state of Ceará during the rainy and dry seasons. The greatest density was in April (median = 12.5 triatomines/site). The greatest abundance was in December, when the insects were more dispersed and the density per site was lower (6 triatomines/site). The nutritional status of females and 5th instar nymphs was increased in July. The rate of T. cruzi infection reached its highest peak in July (10.9%). ELISA revealed that the principal food sources were birds (33.1%), followed by armadillos (18.8%). Food sources were more frequently identified during the rainy season. T. brasiliensis specimens collected in the drought tended to: i) present lower rates of T. cruzi infection and gut content reactivity to tested antisera, ii) have a poorer nutritional status, iii) exhibit lower fecundity, iv) be more dispersed among the studied collection sites, and v) be more abundant and easily collected in the surface of the rocks, possibly reflecting an increased searching for blood meals. Such findings underscore epidemiological concerns and allow inferences about the season when triatomines can more frequently invade the peridomestic environment in search of food and recolonize artificial structures.
Menino, João Filipe; Saraiva, Margarida; Gomes-Rezende, Jéssica; Sturme, Mark; Pedrosa, Jorge; Castro, António Gil; Ludovico, Paula; Goldman, Gustavo H.; Rodrigues, Fernando
Conidia/mycelium-to-yeast transition of Paracoccidioidesbrasiliensis is a critical step for the establishment of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Thus, knowledge of the factors that mediate this transition is of major importance for the design of intervention strategies. So far, the only known pre-requisites for the accomplishment of the morphological transition are the temperature shift to 37°C and the availability of organic sulfur compounds. In this study, we investigated the auxotrophic nature to organic sulfur of the yeast phase of Paracoccidioides, with special attention to P. brasiliensis species. For this, we addressed the role of SconCp, the negative regulator of the inorganic sulfur assimilation pathway, in the dimorphism and virulence of this pathogen. We show that down-regulation of SCONC allows initial steps of mycelium-to-yeast transition in the absence of organic sulfur compounds, contrarily to the wild-type fungus that cannot undergo mycelium-to-yeast transition under such conditions. However, SCONC down-regulated transformants were unable to sustain yeast growth using inorganic sulfur compounds only. Moreover, pulses with inorganic sulfur in SCONC down-regulated transformants triggered an increase of the inorganic sulfur metabolism, which culminated in a drastic reduction of the ATP and NADPH cellular levels and in higher oxidative stress. Importantly, the down-regulation of SCONC resulted in a decreased virulence of P. brasiliensis, as validated in an in vivo model of infection. Overall, our findings shed light on the inability of P. brasiliensis yeast to rely on inorganic sulfur compounds, correlating its metabolism with cellular energy and redox imbalances. Furthermore, the data herein presented reveal SconCp as a novel virulence determinant of P. brasiliensis. PMID:24066151
Stevens, R.L.; Lee, T.D.G.; Seldin, D.C.; Austen, K.F.; Befus, A.D.; Bienenstock, J.
Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of (/sup 35/S) sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the /sup 35/S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The isolated proteoglycans were of approx. 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched populations of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leumekia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans are all highly sulfated, protease-resistant proteoglycans.
Muñoz, José F.; Desjardins, Christopher A.; Gallo, Juan E.; Sykes, Sean; Sakthikumar, Sharadha; Misas, Elizabeth; Whiston, Emily A.; Bagagli, Eduardo; Soares, Celia M. A.; Teixeira, Marcus de M.; Taylor, John W.; Clay, Oliver K.; McEwen, Juan G.
ABSTRACT The Paracoccidioides genus includes two species of thermally dimorphic fungi that cause paracoccidioidomycosis, a neglected health-threatening human systemic mycosis endemic to Latin America. To examine the genome evolution and the diversity of Paracoccidioides spp., we conducted whole-genome sequencing of 31 isolates representing the phylogenetic, geographic, and ecological breadth of the genus. These samples included clinical, environmental and laboratory reference strains of the S1, PS2, PS3, and PS4 lineages of P. brasiliensis and also isolates of Paracoccidioides lutzii species. We completed the first annotated genome assemblies for the PS3 and PS4 lineages and found that gene order was highly conserved across the major lineages, with only a few chromosomal rearrangements. Comparing whole-genome assemblies of the major lineages with single-nucleotide polymorphisms (SNPs) predicted from the remaining 26 isolates, we identified a deep split of the S1 lineage into two clades we named S1a and S1b. We found evidence for greater genetic exchange between the S1b lineage and all other lineages; this may reflect the broad geographic range of S1b, which is often sympatric with the remaining, largely geographically isolated lineages. In addition, we found evidence of positive selection for the GP43 and PGA1 antigen genes and genes coding for other secreted proteins and proteases and lineage-specific loss-of-function mutations in cell wall and protease genes; these together may contribute to virulence and host immune response variation among natural isolates of Paracoccidioides spp. These insights into the recent evolutionary events highlight important differences between the lineages that could impact the distribution, pathogenicity, and ecology of Paracoccidioides. IMPORTANCE Characterization of genetic differences between lineages of the dimorphic human-pathogenic fungus Paracoccidioides can identify changes linked to important phenotypes and guide the
Arruda, Denise C; Matsuo, Alisson L; Silva, Luiz S; Real, Fernando; Leitão, Natanael P; Pires, Jhon H S; Caires, Antonio Carlos F; Garcia, Daniel M; Cunha, Fernanda F M; Puccia, Rosana; Longo, Larissa V G
Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 μg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses.
Arruda, Denise C.; Matsuo, Alisson L.; Silva, Luiz S.; Real, Fernando; Leitão, Natanael P.; Pires, Jhon H. S.; Caires, Antonio Carlos F.; Garcia, Daniel M.; Cunha, Fernanda F. M.; Longo, Larissa V. G.
Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 μg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses. PMID:26349827
Queiroz Júnior, Luiz de Pádua; de Camargo, Zoilo Pires; Tadano, Tomoko; Rodrigues, Anderson Messias; Takarara, Doracilde Terumi; Gegembauer, Gregory; Araujo, Leticia Mendes; Hahn, Rosane Christine
Clinical Paracoccidioides spp. isolates from patients with paracoccidioidomycosis (PCM) in Mato Grosso, Brazil exhibit different patterns of serologic reactivity. The results observed for reactions of radial immunodiffusion against the commonly used exoantigens containing a 43-kDa glycoprotein (gp43) suggest that this fungus exhibits major antigenic variability by geographic region. There is a phylogenetic gap between Paracoccidioides spp. isolates among different regions of Latin America. In particular, those from the central region of Brazil (i.e. Mato Grosso state) exhibit a lower rate of genetic similarity. We aimed at investigating the phylogenetic classification of clinical isolates of Paracoccidioides spp. in Central Brazil and the different antigenic profiles that produce. Exoantigens were obtained from five clinical isolates: two P. brasiliensis (Pb166 and Pb2880) and three P. lutzii (PL2875, PL9840, and PL2912). The protein/glycoprotein profiles of P. lutzii exoantigens were different from each other. Isolate PL9840 exhibited the most distinct bands, and isolates PL2875 and PL2912 exhibited more diffuse bands and a very intense band between 50 and 60 kDa. P. brasiliensis isolates had similar protein profiles, exhibiting a low-intensity band at 220 kDa and a diffuse band between 50 and 60 kDa. P. lutzii isolates exhibit high species-specific antigen variability, which we have already been assessed in proteomic studies.
Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida
Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516
Immune modulation by helminth (worm) parasites could protect the host against autoimmune diseases. We report that the parasitic nematode Nippostrongylus brasiliensis induces changes in the expression of antimicrobial peptides that are associated with marked microbial composition shifts, including re...
Silvana dos Santos, Suelen; Ferreira, Karen Spadari; Almeida, Sandro Rogerio
Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response. PMID:21611175
Lima, Patrícia de Sousa; Casaletti, Luciana; Bailão, Alexandre Melo; de Vasconcelos, Ana Tereza Ribeiro; Fernandes, Gabriel da Rocha; Soares, Célia Maria de Almeida
Background The genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis. Methodology/Principal Findings The fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MSE) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages. Conclusions/Significance For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding
Conde, C; Melendro, E I; Fresán, M; Ortiz-Ortiz, L
The capacity of Nocardia brasiliensis to induce mycetoma in BALB/c mice in the absence of adjuvants was studied. Whether the virulence of N. brasiliensis varied in the different phases of its growth cycle was also determined. The results showed that N. brasiliensis suspended in 0.15 M NaCl and injected into the footpads of mice were able to induce mycetoma after only 14 days of infection, as evidenced by histological studies. Data are also presented indicating that the virulence of N. brasiliensis did not vary during the different phases of its growth curve. The differences in virulence reported between N. brasiliensis and other nocardiae are discussed and explained in terms of several variables in the experimental designs used: among these variables are mouse genotype, route of inoculation, and model for determining virulence. Images PMID:7152671
Farmer, S. G.; Laniyonu, A. A.
Infection of rats with the nematode N. brasiliensis caused non-specific increases in maximum response of isolated intestine to acetylcholine and 5-hydroxytryptamine (5-HT), and a specific subsensitivity to 5-HT. Intestinal levels of 5-HT, measured fluorimetrically, increased approximately 2 fold during infection. Treatment of infected rats with parachlorophenylalanine (PCPA) depleted the gut of 5-HT, and prevented the specific subsensitivity to the amine but not the increases in maximum response. Depletion of intestinal 5-HT did not prevent the immune expulsion of the parasites. It is concluded that the specific subsensitivity of the gut is due to the elevated levels of 5-HT during infection, but that the increased maximum responses are due to some other factor. Further, the lack of effect of PCPA on parasite rejection casts doubt on the proposed role of 5-HT in this process. PMID:6236863
This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases. PMID:25180029
Almeida, Carlos Eduardo; Faucher, Leslie; Lavina, Morgane; Costa, Jane; Harry, Myriam
We used an individual-based molecular multisource approach to assess the epidemiological importance of Triatoma brasiliensis collected in distinct sites and ecotopes in Rio Grande do Norte State, Brazil. In the semi-arid zones of Brazil, this blood sucking bug is the most important vector of Trypanosoma cruzi—the parasite that causes Chagas disease. First, cytochrome b (cytb) and microsatellite markers were used for inferences on the genetic structure of five populations (108 bugs). Second, we determined the natural T. cruzi infection prevalence and parasite diversity in 126 bugs by amplifying a mini-exon gene from triatomine gut contents. Third, we identified the natural feeding sources of 60 T. brasiliensis by using the blood meal content via vertebrate cytb analysis. Demographic inferences based on cytb variation indicated expansion events in some sylvatic and domiciliary populations. Microsatellite results indicated gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations, which threatens vector control efforts because sylvatic population are uncontrollable. A high natural T. cruzi infection prevalence (52–71%) and two parasite lineages were found for the sylvatic foci, in which 68% of bugs had fed on Kerodon rupestris (Rodentia: Caviidae), highlighting it as a potential reservoir. For peridomiciliary bugs, Galea spixii (Rodentia: Caviidae) was the main mammal feeding source, which may reinforce previous concerns about the potential of this animal to link the sylvatic and domiciliary T. cruzi cycles. PMID:26891047
Almeida, Carlos Eduardo; Faucher, Leslie; Lavina, Morgane; Costa, Jane; Harry, Myriam
We used an individual-based molecular multisource approach to assess the epidemiological importance of Triatoma brasiliensis collected in distinct sites and ecotopes in Rio Grande do Norte State, Brazil. In the semi-arid zones of Brazil, this blood sucking bug is the most important vector of Trypanosoma cruzi--the parasite that causes Chagas disease. First, cytochrome b (cytb) and microsatellite markers were used for inferences on the genetic structure of five populations (108 bugs). Second, we determined the natural T. cruzi infection prevalence and parasite diversity in 126 bugs by amplifying a mini-exon gene from triatomine gut contents. Third, we identified the natural feeding sources of 60 T. brasiliensis by using the blood meal content via vertebrate cytb analysis. Demographic inferences based on cytb variation indicated expansion events in some sylvatic and domiciliary populations. Microsatellite results indicated gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations, which threatens vector control efforts because sylvatic population are uncontrollable. A high natural T. cruzi infection prevalence (52-71%) and two parasite lineages were found for the sylvatic foci, in which 68% of bugs had fed on Kerodon rupestris (Rodentia: Caviidae), highlighting it as a potential reservoir. For peridomiciliary bugs, Galea spixii (Rodentia: Caviidae) was the main mammal feeding source, which may reinforce previous concerns about the potential of this animal to link the sylvatic and domiciliary T. cruzi cycles.
Sperandio, Felipe Fornias; Fernandes, Gisele Pesquero; Mendes, Ana Carolina Silvério Cerqueira; Bani, Giulia Maria de Alencar Castro; Calich, Vera Lucia Garcia; Burger, Eva
Paracoccidioidomycosis (PCM) is a systemic fungal infection, endemic in Brazil, that leads to severe morbidity and even mortality if not correctly treated. Patients may respond differently to PCM depending on the pattern of the acquired immune response developed. The onset of protective immune response is notably mediated by neutrophils (PMN) that play an important role through directly killing the fungi and also by interacting with other cell types to modulate the acquired protective immune response that may follow. In that way, this study aimed to present and compare different experimental models of PCM (intraperitoneal and subcutaneous) regarding PMN production and maturation inside femoral bone marrow and also PMN infiltration in peritoneal and subcutaneous exudates of resistant and susceptible mice. We also assessed the fungal colony forming units and the levels of soluble inflammatory mediators (LTB4, KC, IFN-γ, GM-CSF, and IL-10) inside subcutaneous air-pouches to compare the efficiency of the PMN present at this site in relation to the two main neutrophil functions: initial lysis of the invading pathogen and modulation of the acquired immune response. P. brasiliensis inoculated intraperitoneally was able to disseminate to the bone marrow of susceptible mice, causing a more marked alteration of PMN production and maturation than that observed after resistant mice infection by the same route. Subcutaneous air-pouch inoculation of P. brasiliensis elicited a controlled and limited infection that produced a PMN-rich exudate, thus favoring the study of the interaction between the fungus and the neutrophils. Susceptible mice produced higher numbers of PMN; however, these cells were less effective in killing the fungi. Inflammatory cytokines were more pronounced in resistant mice, which supports their PCM raised resistance. PMID:26819497
Katona, I M; Urban, J F; Finkelman, F D
Recently, it has been reported that in SJA/9 mice infected with Nippostrongylus brasiliensis there are increased numbers of lymphoid cells positive for surface IgM and IgE (sIgM+ and sIgE+) even though they fail to secrete IgE, that both the sIgM and sIgE on these cells are intrinsic, and that there has been no deletion of genes for the Ig heavy chain constant region in these cells. These observations support a nondeletional model for Ig isotype switching. We have now reexamined the nature of sIgE on sIgE+ spleen and mesenteric lymph node cells of N. brasiliensis-infected SJA/9 mice, and the following observations lead us to believe that this sIgE is cytophilic rather than intrinsic: (i) Only approximately 50% of the N. brasiliensis-infected SJA/9 mice have detectable percentages of sIgE+ lymphoid cells. All mice with detectable sIgE+ lymphocytes have lymphocytes positive for intracytoplasmic IgE (cIgE+) and secrete IgE in vitro, while cIgE+ cells and IgE secretion are absent from N. brasiliensis-infected SJA/9 mice that lack sIgE+ cells. (ii) SJA/9 B lymphocytes have receptors for IgE: expression of these receptors is increased in N. brasiliensis-infected mice that have sIgE+ lymphocytes, but not in infected SJA/9 mice that lack sIgE+ lymphocytes. (iii) Treatment of sIgM+ sIgD+ sIgE+ cells for 1 min with dilute acid removes most sIgE but does not affect expression of sIgM or sIgD. (iv) The removal of mouse IgE from sIgE+ B cells facilitates the binding of exogenous rat IgE. (v) The small amount of sIgE that is reexpressed during a period of in vitro culture after acid treatment is blocked by inclusion of exogenous rat IgE in the culture medium. These observations show that most sIgM+ sIgE+ B cells in N. brasiliensis-infected SJA/9 mice do not express intrinsic sIgE; thus studies using these cells to determine mechanisms of Ig isotype switching are inconclusive. PMID:3155861
Stading, Benjamin; Osorio, Jorge E.; Velasco-Villa, Andres; Smotherman, Michael; Kingstad-Bakke, Brock; Rocke, Tonie E.
Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value as insectivores and plant dispersers, but their role as reservoirs of zoonotic diseases has received more attention in the last decade. With the goal of managing disease in free-ranging bats, we tested modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) as potential vaccine vectors in the Brazilian Free-tailed bat (Tadarida brasiliensis), using biophotonic in vivo imaging and immunogenicity studies. Animals were administered recombinant poxviral vectors expressing the luciferase gene (MVA-luc, RCN-luc) through oronasal (ON) or intramuscular (IM) routes and subsequently monitored for bioluminescent signal indicative of viral infection. No clinical illness was noted after exposure to any of the vectors, and limited luciferase expression was observed. Higher and longer levels of expression were observed with the RCN-luc construct. When given IM, luciferase expression was limited to the site of injection, while ON exposure led to initial expression in the oral cavity, often followed by secondary replication at another location, likely the gastric mucosa or gastric associated lymphatic tissue. Viral DNA was detected in oral swabs up to 7 and 9 days post infection (dpi) for MVA and RCN, respectively. While no live virus was detected in oral swabs from MVA-infected bats, titers up to 3.88 x 104 PFU/ml were recovered from oral swabs of RCN-infected bats. Viral DNA was also detected in fecal samples from two bats inoculated IM with RCN, but no live virus was recovered. Finally, we examined the immunogenicity of a RCN based rabies vaccine (RCN-G) following ON administration. Significant rabies neutralizing antibody titers were detected in the serum of immunized bats using the rapid fluorescence focus inhibition test (RFFIT). These studies highlight the safety and immunogenicity of attenuated poxviruses and their potential use as vaccine vectors in bats.
Stading, Ben R; Osorio, Jorge E; Velasco-Villa, Andres; Smotherman, Michael; Kingstad-Bakke, Brock; Rocke, Tonie E
Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value as insectivores and plant dispersers, but their role as reservoirs of zoonotic diseases has received more attention in the last decade. With the goal of managing disease in free-ranging bats, we tested modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) as potential vaccine vectors in the Brazilian Free-tailed bat (Tadarida brasiliensis), using biophotonic in vivo imaging and immunogenicity studies. Animals were administered recombinant poxviral vectors expressing the luciferase gene (MVA-luc, RCN-luc) through oronasal (ON) or intramuscular (IM) routes and subsequently monitored for bioluminescent signal indicative of viral infection. No clinical illness was noted after exposure to any of the vectors, and limited luciferase expression was observed. Higher and longer levels of expression were observed with the RCN-luc construct. When given IM, luciferase expression was limited to the site of injection, while ON exposure led to initial expression in the oral cavity, often followed by secondary replication at another location, likely the gastric mucosa or gastric associated lymphatic tissue. Viral DNA was detected in oral swabs up to 7 and 9 days post infection (dpi) for MVA and RCN, respectively. While no live virus was detected in oral swabs from MVA-infected bats, titers up to 3.88 x 10(4) PFU/ml were recovered from oral swabs of RCN-infected bats. Viral DNA was also detected in fecal samples from two bats inoculated IM with RCN, but no live virus was recovered. Finally, we examined the immunogenicity of a RCN based rabies vaccine (RCN-G) following ON administration. Significant rabies neutralizing antibody titers were detected in the serum of immunized bats using the rapid fluorescence focus inhibition test (RFFIT). These studies highlight the safety and immunogenicity of attenuated poxviruses and their potential use as vaccine vectors in bats.
Silva, Ayrles F B; Matos, Mayara P V; Ralph, Maria T; Silva, Daiane L; de Alencar, Nylane M; Ramos, Márcio V; Lima-Filho, José V
The immunomodulatory properties of mannose-binding lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) were investigated comparatively in a model of Salmonella infection. The lectins were intraperitoneally (i.p.) administered to mice daily for three days before the bacterial challenge with Salmonella enterica Ser. Typhimurium (0.2 mL i.p.; 10(7) CFU/mL). In vivo assays have shown that both lectins induced a significant leukocyte infiltration into the peritoneal cavity of uninfected mice, which was higher in the CFL group 3 days post-infection. Total and differential cell counts in the bloodstreams have shown uninfected animals pretreated with ConBr and CFL exhibited accentuated lymphopenia. Conversely, there was an increasing population of lymphocytes following 3 days post-infection in mice pretreated with both lectins. In addition, the bacterial burden was significantly reduced into the peritoneal cavity, bloodstreams, spleen and the liver in these mice. The lectins did not induce the release of pro- or anti-inflammatory cytokines into the peritoneal fluid of uninfected animals. However, following infection, the release of TNF-α and IL-10 in the peritoneal fluid were down-regulated in mice pretreated with both lectins whereas IL-1 was only reduced in mice pretreated with ConBr. Uninfected animals pretreated with CFL exhibited high nitric oxide (NO) content in the peritoneal fluid, which was decreased after infection in comparison to ConBr group. The lectins did not alter the serum levels of NO in uninfected mice but treatments with ConBr significantly reduced the NO content in infected animals in comparison to CFL group 24h after the bacterial challenge. Survival experiments have shown survival rates ranging from 70% to 100% in mice that received CFL or ConBr. On the other hand, untreated mice (PBS group) died 1-6 days after infection. We conclude that ConBr and CFL are prospective phytotherapeutics capable of modulate the cascade of pro
da Silva, Roberta Peres; Heiss, Christian; Black, Ian; ...
Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) andmore » α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.« less
Oliveira, Lucas Nojosa; Casaletti, Luciana; Báo, Sônia Nair; Borges, Clayton Luiz; de Sousa Lima, Patrícia; de Almeida Soares, Célia Maria
Paracoccidioidomycosis is an endemic disease in Latin America, caused by thermo dimorphic fungi of the genus Paracoccidioides. Although previous proteome analyses of Paracoccidioides spp. have been carried out, the nuclear subproteome of this pathogen has not been described. In this way, we aimed to characterize the nuclear proteome of Paracoccidioides species, in the yeast form. For that, yeast cells were disrupted and submitted to cell fractionation. The purity of the nuclear fraction was confirmed by fluorescence and electron microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 867 proteins. In order to support our enrichment method for nuclear proteins, bioinformatics analysis were applied that allowed the identification of 281 proteins with nuclear localization. The analysis revealed proteins related to DNA maintenance, gene expression, synthesis and processing of messenger and ribosomal RNAs, likewise proteins of nuclear-cytoplasmic traffic. It was also possible to detect some proteins that are poorly expressed, like transcription factors involved in important roles such as resistance to abiotic stress, sporulation, cellular growth and DNA and chromatin maintenance. This is the first descriptive nuclear proteome of Paracoccidioides spp. that can be useful as an important platform base for fungi-specific nuclear processes.
Rammaert, B; Gamaletsou, M N; Zeller, V; Elie, C; Prinapori, R; Taj-Aldeen, S J; Roilides, E; Kontoyiannis, D P; Brause, B; Sipsas, N V; Walsh, T J; Lortholary, O
The objective of this investigation was to review the clinical manifestations, management, and outcome of osteoarticular infections caused by dimorphic fungi. We exhaustively reviewed reports of bone and joint infections caused by dimorphic fungi published between 1970 and 2012. Underlying conditions, microbiological features, histological characteristics, clinical manifestations, antifungal therapy, and outcome were analyzed in 222 evaluable cases. Among 222 proven cases (median age 41 years [interquartile range (IQR) 26-57]), 73 % had no predisposing condition. Histopathology performed in 128 (57 %) cases and culture in 170 confirmed diagnosis in 63 % and 98 % of the cases, respectively. Diagnosis was obtained from an extra-osteoarticular site in 16 cases. The median diagnostic time was 175 days (IQR 60-365). Sporothrix schenckii was the most frequent pathogen (n = 84), followed by Coccidioides immitis (n = 47), Blastomyces dermatitidis (n = 44), Histoplasma capsulatum (n = 18), Paracoccidioides brasiliensis (n = 16), and Penicillium marneffei (n = 13). Arthritis occurred in 87 (58 %) cases and osteomyelitis in 64 (42 %), including 19 vertebral osteomyelitis. Dissemination was reported in 123 (55 %) cases. Systemic antifungal agents were used in 216 (97 %) patients and in combination with surgery in 129 (60 %). Following the Infectious Diseases Society of America (IDSA) guidelines, a successful initial medical strategy was observed in 97/116 (84 %) evaluable cases. The overall mortality was 6 %, and was highest for P. marneffei (38.5 %). This study demonstrates that dimorphic osteoarticular infections have distinctive clinical presentations, occur predominantly in apparently immunocompetent patients, develop often during disseminated disease, and may require surgical intervention.
Sperandio, F. F.; Bani, G. M. A. C.; Mendes, A. C. S. C.; Brigagão, M. R. P. L.; Santos, G. B.; Malaquias, L. C. C.; Chavasco, J. K.; Verinaud, L. M.; Burger, E.
Polymorphonuclear neutrophils (PMN) participate in an active way in the innate immunity developed after the fungal infection paracoccidioidomycosis (PCM). Nevertheless, the sole participation of neutrophils is not sufficient to eradicate PCM`s pathogenic fungus: Paracoccidioides brasiliensis (Pb). In that way, we aimed to develop a treatment capable of stimulating PMN to the site of injury through low-level laser therapy (LLLT). (LLLT) is safe to use and has not been linked to microorganism resistance so far; in addition, based on previous studies we understand that LLLT may be useful to treat several medical conditions through the stimulation and activation of certain types of cells. This brief review is based on the novel attempt of activating PMN against a fungal infection.
Lima, Patrícia de Sousa; Chung, Dawoon; Bailão, Alexandre Melo; Cramer, Robert A.; Soares, Célia Maria de Almeida
Background Hypoxic microenvironments are generated during fungal infection. It has been described that to survive in the human host, fungi must also tolerate and overcome in vivo microenvironmental stress conditions including low oxygen tension; however nothing is known how Paracoccidioides species respond to hypoxia. The genus Paracoccidioides comprises human thermal dimorphic fungi and are causative agents of paracoccidioidomycosis (PCM), an important mycosis in Latin America. Methodology/Principal Findings In this work, a detailed hypoxia characterization was performed in Paracoccidioides. Using NanoUPLC-MSE proteomic approach, we obtained a total of 288 proteins differentially regulated in 12 and 24 h of hypoxia, providing a global view of metabolic changes during this stress. In addition, a functional characterization of the homologue to the most important molecule involved in hypoxia responses in other fungi, the SREBP (sterol regulatory element binding protein) was performed. We observed that Paracoccidioides species have a functional homologue of SREBP, named here as SrbA, detected by using a heterologous genetic approach in the srbA null mutant in Aspergillus fumigatus. Paracoccidioides srbA (PbsrbA), in addition to involvement in hypoxia, is probable involved in iron adaptation and azole drug resistance responses. Conclusions/Significance In this study, the hypoxia was characterized in Paracoccidioides. The first results can be important for a better understanding of the fungal adaptation to the host and improve the arsenal of molecules for the development of alternative treatment options in future, since molecules related to fungal adaptation to low oxygen levels are important to virulence and pathogenesis in human pathogenic fungi. PMID:26659387
Coelho, Paulo S R; Im, Hogune; Clemons, Karl V; Snyder, Michael P; Stevens, David A
We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine.
Deepe, George S
Many challenges confront the development of fungal vaccines for humans including differences in host susceptibility, varied pathogenic mechanisms employed by the different species of fungi and mechanisms of host resistance. Hence, no single antigen can be expected to serve as a pan fungal vaccine. Instead, it is likely that progress for fungal vaccines will have to be made at the level of each individual organism. In recent years, tremendous strides have been made in understanding the immunopathogenesis of medically important fungal infections and identifying putative vaccine candidates. Such discoveries will facilitate the introduction of fungal vaccines into the therapeutic armamentarium of clinicians. The fungi under discussion in this review include Candida spp., Aspergillus spp., Cryptococcus neoformans, Coccidioides spp., Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Pneumocystis jirovecii.
da Silva, Roberta Peres; Heiss, Christian; Black, Ian; Azadi, Parastoo; Gerlach, Jared Q.; Travassos, Luiz R.; Joshi, Lokesh; Kilcoyne, Michelle; Puccia, Rosana
Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.
Cattana, Maria Emilia; Tracogna, Maria Fernanda; Marques, Isabel; Rojas, Florencia; Fernández, Mariana; de Los Ángeles Sosa, María; Mussin, Javier; Giusiano, Gustavo
Paracoccidioidomycosis is an endemic mycosis caused by Paracoccidioides species limited to Latin America arising with the chronic form in 90% of cases. The capacity of microorganisms to form biofilms is considered of great importance medical since can contribute to the persistence and to the chronic state of the diseases. The ability of Paracoccidioides to form biofilm has been demonstrated in vitro. In our study, for the first time we have observed this capability in vivo on a vascular prosthesis using scanning electron microscope showing a dense network of Paracoccidioides yeasts covered by an extracellular matrix.
Chaudhary, Anshu; Goswami, Urvashi; Singh, Hridaya Shanker
Mus musculus (Rodentia: Muridae) has generally been infected with a rodent hookworm Nippostrongylus brasiliensis. In this report, we present morphological and molecular identification of N. brasiliensis by light and scanning electron microscopy and PCR amplification of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and the protein sequences encoded by cox1 gene, respectively. Despite the use of N. brasiliensis in many biochemistry studies from India, their taxonomic identification was not fully understood, especially at the species level, and no molecular data is available in GenBank from India. Sequence analysis of cox1 gene in this study revealed that the present specimen showed close identity with the same species available in GenBank, confirming that the species is N. brasiliensis. This study represents the first record of molecular identification of N. brasiliensis from India and the protein structure to better understand the comparative phylogenetic characteristics. PMID:28095659
This is the first recorded case of rabies in the insectivorous bat Tadarida brasiliensis in the State of S. Paulo, Southeastern Brazil. The infected bat was found in the afternoon while hanging on the internal wall of an urban building. This observation reinforces the notion as to the caution one must exercise regarding bats found in unusual situations.
Cano, L E; Brummer, E; Stevens, D A; Restrepo, A
Conidia ingested by resident macrophages had an enhanced percentage of transformation to yeast cells compared with those in culture medium without macrophages. The yeast cells subsequently grew intracellularly by budding. Macrophages treated with cytokines from antigen-stimulated spleen cells from immunized mice significantly inhibited transformation of ingested conidia. PMID:1563800
Cano, L E; Brummer, E; Stevens, D A; Restrepo, A
Conidia ingested by resident macrophages had an enhanced percentage of transformation to yeast cells compared with those in culture medium without macrophages. The yeast cells subsequently grew intracellularly by budding. Macrophages treated with cytokines from antigen-stimulated spleen cells from immunized mice significantly inhibited transformation of ingested conidia.
Weber, Simone Schneider; Parente, Ana Flávia Alves; Borges, Clayton Luiz; Parente, Juliana Alves; Bailão, Alexandre Melo; de Almeida Soares, Célia Maria
Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host. PMID:23272246
do Prado, Renata Silva; Alves, Ricardo Justino; de Oliveira, Cecília Maria Alves; Kato, Lucília; da Silva, Roosevelt Alves; Quintino, Guilherme Oliveira; do Desterro Cunha, Silvio; de Almeida Soares, Célia Maria; Pereira, Maristela
The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments. PMID:24752170
Background The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-β-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress. Results We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment. Conclusions The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus. PMID:24119145
Tomazett, Mariana Vieira; Zanoelo, Fabiana Fonseca; Bailão, Elisa Flávia Cardoso; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida
Abstract Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4. PMID:27560991
Salem-Izacc, S M; Jesuino, R S; Brito, W A; Pereira, M; Felipe, M S; Soares, C M
In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.
Carreto-Binaghi, Laura Elena; Damasceno, Lisandra Serra; Pitangui, Nayla de Souza; Fusco-Almeida, Ana Marisa; Mendes-Giannini, Maria José Soares; Zancopé-Oliveira, Rosely Maria; Taylor, Maria Lucia
Healthcare-associated infections (HAI) are described in diverse settings. The main etiologic agents of HAI are bacteria (85%) and fungi (13%). Some factors increase the risk for HAI, particularly the use of medical devices; patients with severe cuts, wounds, and burns; stays in the intensive care unit, surgery, and hospital reconstruction works. Several fungal HAI are caused by Candida spp., usually from an endogenous source; however, cross-transmission via the hands of healthcare workers or contaminated devices can occur. Although other medically important fungi, such as Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Histoplasma capsulatum, have never been considered nosocomial pathogens, there are some factors that point out the pros and cons for this possibility. Among these fungi, H. capsulatum infection has been linked to different medical devices and surgery implants. The filamentous form of H. capsulatum may be present in hospital settings, as this fungus adapts to different types of climates and has great dispersion ability. Although conventional pathogen identification techniques have never identified H. capsulatum in the hospital environment, molecular biology procedures could be useful in this setting. More research on H. capsulatum as a HAI etiologic agent is needed, since it causes a severe and often fatal disease in immunocompromised patients.
Carreto-Binaghi, Laura Elena; Damasceno, Lisandra Serra; Mendes-Giannini, Maria José Soares; Zancopé-Oliveira, Rosely Maria; Taylor, Maria Lucia
Healthcare-associated infections (HAI) are described in diverse settings. The main etiologic agents of HAI are bacteria (85%) and fungi (13%). Some factors increase the risk for HAI, particularly the use of medical devices; patients with severe cuts, wounds, and burns; stays in the intensive care unit, surgery, and hospital reconstruction works. Several fungal HAI are caused by Candida spp., usually from an endogenous source; however, cross-transmission via the hands of healthcare workers or contaminated devices can occur. Although other medically important fungi, such as Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Histoplasma capsulatum, have never been considered nosocomial pathogens, there are some factors that point out the pros and cons for this possibility. Among these fungi, H. capsulatum infection has been linked to different medical devices and surgery implants. The filamentous form of H. capsulatum may be present in hospital settings, as this fungus adapts to different types of climates and has great dispersion ability. Although conventional pathogen identification techniques have never identified H. capsulatum in the hospital environment, molecular biology procedures could be useful in this setting. More research on H. capsulatum as a HAI etiologic agent is needed, since it causes a severe and often fatal disease in immunocompromised patients. PMID:26106622
Almeida-Paes, Rodrigo; de Oliveira, Manoel Marques Evangelista; Freitas, Dayvison Francis Saraiva; do Valle, Antônio Carlos Francesconi; Zancopé-Oliveira, Rosely Maria; Gutierrez-Galhardo, Maria Clara
Background There have been several recent changes in the taxonomy of Sporothrix schenckii as well as new observations regarding the clinical aspects of sporotrichosis. In this study, we determined the identification of the Sporothrix species associated with both classic and unusual clinical aspects of sporotrichosis observed in the endemic area of sporotrichosis in Rio de Janeiro, Brazil. Methodology/Principal Findings To verify whether S. brasiliensis is associated with clinical manifestations of sporotrichosis, a cross-sectional study was performed in which Sporothrix isolates from 50 patients with different clinical manifestations were analyzed and their isolates were studied by phenotypic and genotypic methods. Data from these patients revealed a distinct clinical picture and therapeutic response in infections caused by Sporothrix brasiliensis (n = 45) compared to patients with S. schenckii sensu stricto (n = 5). S. brasiliensis was associated with disseminated cutaneous infection without underlying disease, hypersensitivity reactions, and mucosal infection, whereas patients with S. schenckii presented with less severe and more often localized disease, similar to the majority of previously described sporotrichosis cases. Interestingly, S. brasiliensis-infected patients overall required shorter durations of itraconazole (median 16 weeks) compared to the individuals with S. schenckii (median 24 weeks). Conclusions/Significance These findings suggest that Sporothrix species are linked to different clinical manifestations of sporotrichosis and that S. brasiliensis is effectively treated with oral itraconazole. PMID:25233227
Rodrigues, Anderson Messias; de Melo Teixeira, Marcus; de Hoog, G Sybren; Schubach, Tânia Maria Pacheco; Pereira, Sandro Antonio; Fernandes, Geisa Ferreira; Bezerra, Leila Maria Lopes; Felipe, Maria Sueli; de Camargo, Zoilo Pires
Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n =3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.
Rodrigues, Anderson Messias; de Melo Teixeira, Marcus; de Hoog, G. Sybren; Schubach, Tânia Maria Pacheco; Pereira, Sandro Antonio; Fernandes, Geisa Ferreira; Bezerra, Leila Maria Lopes; Felipe, Maria Sueli; de Camargo, Zoilo Pires
Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n = 3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil. PMID:23818999
Thomaz, Luciana; García-Rodas, Rocío; Guimarães, Allan J; Taborda, Carlos P; Zaragoza, Oscar; Nosanchuk, Joshua D
Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.
Brilhante, Raimunda Sâmia Nogueira; Rodrigues, Anderson Messias; Sidrim, José Júlio Costa; Rocha, Marcos Fábio Gadelha; Pereira, Sandro Antonio; Gremião, Isabella Dib Ferreira; Schubach, Tânia Maria Pacheco; de Camargo, Zoilo Pires
Sporotrichosis is an important subcutaneous mycosis of humans and animals. Classically, the disease is acquired upon traumatic inoculation of Sporothrix propagules from contaminated soil and plant debris. In addition, the direct horizontal transmission of Sporothrix among animals and the resulting zoonotic infection in humans highlight an alternative and efficient rout of transmission through biting and scratching. Sporothrix brasiliensis is the most virulent species of the Sporothrix schenckii complex and is responsible for the long-lasting outbreak of feline sporotrichosis in Brazil. However, antifungal susceptibility data of animal-borne isolates is scarce. Therefore, this study evaluated the in vitro activity of amphotericin B, caspofungin, itraconazole, voriconazole, fluconazole, and ketoconazole against animal-borne isolates of S. brasiliensis. The susceptibility tests were performed through broth microdilution (M38-A2). The results show the relevant activity of itraconazole, amphotericin B, and ketoconazole against S. brasiliensis, with the following MIC ranges: 0.125-2, 0.125-4 and 0.0312-2 μg/ml, respectively. Caspofungin was moderately effective, displaying higher variation in MIC values (0.25-64 μg/ml). Voriconazole (2-64 μg/ml) and fluconazole (62.5-500 μg/ml) showed low activity against S. brasiliensis strains. This study contributed to the characterization of the in vitro antifungal susceptibility of strains of S. brasiliensis recovered from cats with sporotrichosis, which have recently been considered the main source of human infections.
Trevino-Villarreal, J. Humberto; Vera-Cabrera, Lucio; Valero-Guillén, Pedro L.
Nocardia brasiliensis is a Gram-positive facultative intracellular bacterium frequently isolated from human actinomycetoma. However, the pathogenesis of this infection remains unknown. Here, we used a model of bacterial delipidation with benzine to investigate the role of N. brasiliensis cell wall-associated lipids in experimental actinomycetoma. Delipidation of N. brasiliensis with benzine resulted in complete abolition of actinomycetoma without affecting bacterial viability. Chemical analyses revealed that trehalose dimycolate and an unidentified hydrophobic compound were the principal compounds extracted from N. brasiliensis with benzine. By electron microscopy, the extracted lipids were found to be located in the outermost membrane layer of the N. brasiliensis cell wall. They also appeared to confer acid-fastness. In vitro, the extractable lipids from the N. brasiliensis cell wall induced the production of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and CCL-2 in macrophages. The N. brasiliensis cell wall extractable lipids inhibited important macrophage microbicidal effects, such as tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, phagocytosis, bacterial killing, and major histocompatibility complex class II (MHC-II) expression in response to gamma interferon (IFN-γ). In dendritic cells (DCs), N. brasiliensis cell wall-associated extractable lipids suppressed MHC-II, CD80, and CD40 expression while inducing tumor growth factor β (TGF-β) production. Immunization with delipidated N. brasiliensis induced partial protection preventing actinomycetoma. These findings suggest that N. brasiliensis cell wall-associated lipids are important for actinomycetoma development by inducing inflammation and modulating the responses of macrophages and DCs to N. brasiliensis. PMID:22851755
Salinas-Carmona, M C; Castro-Corona, M A; Sepúlveda-Saavedra, J; Perez, L I
We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained. PMID:9067645
Salinas-Carmona, M C; Castro-Corona, M A; Sepúlveda-Saavedra, J; Perez, L I
We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.
Mendoza, Leonel; Belone, Andréa F. F.; Vilela, Raquel; Rehtanz, Manuela; Bossart, Gregory D.; Reif, John S.; Fair, Patricia A.; Durden, Wendy N.; St. Leger, Judy; Travassos, Luiz R.; Rosa, Patricia S.
Antibodies in the sera of patients with lacaziosis recognized an ∼193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera. PMID:17959822
Méndez-Tovar, L J; Mondragón-González, R; Manzano-Gayosso, P; López-Martínez, R; Hernández-Hernández, F; Bonifaz, A; Anides Fonseca, A; Araiza, J; Vega-López, F
Considering that some authors have reported an increasing of some immunoglobulins in actinomycetoma patients, in this study we propose to determine differential production of IgG1, IgG2, IgG3, IgG4 and IgGM in 25 patients with actinomycetoma and 25 healthy individuals from a mycetoma endemic area. Immunoglobulins were determined by ELISA technique. To sensibilize the plates, six Nocardia brasiliensis antigens were used: a crude antigen denominated NB and five derivatives (NB2, NB4, NB6, NB8 and NB10) obtained by their isoelectric point. Results showed that all IgG subclasses were higher in the patients' sera than in control sera, with a maximal difference to IgG3 and IgG4. To the latter subclass, six antigens were highly reactives. IgM levels were similar in both groups. As it occurs in other infections, in the actinomycetoma pathogenesis probably participate the increase or deficiency of a determined immunoglobulin class, as well as the relationship between different subclasses.
Prado, Renata S.; Bailão, Alexandre M.; Silva, Lívia C.; de Oliveira, Cecília M. A.; Marques, Monique F.; Silva, Luciano P.; Silveira-Lacerda, Elisângela P.; Lima, Aliny P.; Soares, Célia M.; Pereira, Maristela
The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MSE. The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied. PMID:26150808
Casaletti, L; Lima, P S; Oliveira, L N; Borges, C L; Báo, S N; Bailão, A M; Soares, C M A
The genus Paracoccidioides is composed of thermal dimorphic fungi, causative agents of paracoccidioidomycosis, one of the most frequent systemic mycoses in Latin America. Mitochondria have sophisticated machinery for ATP production, which involves metabolic pathways such as citric acid and glyoxylate cycles, electron transport chain and oxidative phosphorylation. In addition, this organelle performs a variety of functions in the cell, working as an exceptional metabolic signalling centre that contributes to cellular stress responses, as autophagy and apoptosis in eukaryotic organisms. The aim of this work was to perform a descriptive proteomic analysis of mitochondria in Paracoccidioides lutzii yeast cells. After mitochondria fractionation, samples enriched in mitochondrial proteins were digested with trypsin and analysed using a NanoUPLC-MS(E) system (Waters Corporation, Manchester, UK). Ours results revealed that the established protocol for purification of mitochondria was very effective for P. lutzii, and 298 proteins were identified as primarily mitochondrial, in our analysis. To our knowledge, this is the first compilation of mitochondrial proteins from P. lutzii, to date. Copyright © 2016 John Wiley & Sons, Ltd.
Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria
Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575
Xavier, Melissa O; Pasqualotto, Alessandro C; Cardoso, Isabel Cristina E; Severo, Luiz Carlos
Cross-reactivity in the Platelia Aspergillus enzyme immunoassay was evaluated using 120 sera from patients with paracoccidioidomycosis, histoplasmosis, and cryptococcosis. At a cutoff value of 0.5, positivity rates were 50%, 67%, and 50%, respectively. The implications for these findings are discussed.
Ishida, Kelly; de Castro, Rafaela Alves; Borba Dos Santos, Luana Pereira; Quintella, Leonardo Pereira; Lopes-Bezerra, Leila M; Rozental, Sonia
Sporothrix brasiliensis is a highly virulent member of the S. schenckii complex, which is responsible for the emergence of the epidemic sporotrichosis in southeastern Brazil over the last two decades. There are no in vivo studies on the sensitivity of S. brasiliensis to the therapeutic regimens used to treat sporotrichosis. Here, we evaluated the efficacy and safety of antifungal treatments against S. brasiliensis using a murine model of disseminated sporotrichosis. In vitro, S. brasiliensis yeasts were sensitive to low concentrations of amphotericin B-deoxycholate (AMB-d) and itraconazole (ITZ), the latter having greater selectivity toward the fungus. The following treatment regimens were tested in vivo: intravenous AMB-d for 7 days post-infection (p.i.), oral ITZ for up to 30 days p.i., and AMB-d followed by ITZ (AMB-d/ITZ). AMB-d and AMB-d/ITZ led to 100% survival of infected mice at the end of the 45-day experimental period. Although all treatments extended mice survival, only AMB-d and AMB-d/ITZ significantly reduced fungal load in all organs, but AMB-d/ITZ led to a more consistent decrease in overall fungal burden. No treatment increased the levels of serum toxicity biomarkers. Taken together, our results indicate that AMB-d/ITZ is the best therapeutic option for controlling disseminated sporotrichosis caused by S. brasiliensis.
Almeida-Paes, Rodrigo; Bailão, Alexandre Melo; Pizzini, Cláudia Vera; Reis, Rosani Santos; Soares, Célia Maria de Almeida; Peralta, José Mauro; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely Maria
Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.
Meester, Irene; Rosas-Taraco, Adrian Geovanni; Salinas-Carmona, Mario Cesar
Foamy cells have been described in various infectious diseases, for example in actinomycetoma induced by Nocardia brasiliensis. These cells are generally considered to be macrophages, although they present dendritic cell (DC)-specific surface markers. In this study, we determined and confirmed the lineage of possible precursors of foamy cells in vitro and in vivo using an experimental actinomycetoma model in BALB/c mice. Bone marrow-derived macrophages (BMDM) or DC (BMDC) were infected in vitro with N. brasiliensis or labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Both, macrophages and DC, differentiated into foamy cells after in vitro infection. CFSE-labeled BMDM or BMDC were tested for phagocytosis and CD11c/CD11b receptors markers expression before being transferred into the actinomycetoma lesion site of infected mice. In vivo studies showed that BMDM and BMDC were traced at the site where foamy cells are present in the experimental actinomycetoma. Interestingly, many of the transferred BMDM and BMDC were stained with the lipid-droplet fluorophore Nile Red. In conclusion, macrophages and DC cells can be differentiated into foamy cells in vitro and in vivo during N. brasiliensis infection. PMID:24936860
Cermehol, Julman R; Alvarado, Primavera; Mendoza, Mireya; Herndndez, Isabel; Cuestal, De
Broth microdilution, the reference method recommended by the Clinical Laboratory Standards Institute (CLSI), is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this work, in vitro susceptibility of the Paracoccidioides complex (n=19) to systemic antifungals: amphotericin B, 5-flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole and caspofungin, was evaluated using the microdilution method (Document M27-A3, M27-S3), with some modifications such as: culture time in Sabouraud dextrose agar (7-10 days), RPMI 1640 medium supplemented with 2% glucose and the incubation time (7, 8 and 18 days). The sensitivity in vitro was variable; the majority of Paracoccidioides isolates was susceptible to ketoconazol (73.7%), followed by voriconazole (68.4%), itraconazole (63.1%), amphotericin B (52.6%), fluconazole (47.4%), 5-flucytosine (42.1%) and caspofungin (5%). The overall resistance was mainly to caspofungin (94.7%), followed by 5-flucytosine (52.6%) and amphotericin B (47.4%). Fifty-three percent of the isolates were susceptible-dose dependent to fluconazole followed by itraconazole (15.7%) and 5-fluorocytosine (5.3%). Amphotericin B, itraconazole and voriconazole were the most potent antifungal drugs against Paracoccidioides spp (CMI: 0.03-1 microg/mL). Based on these results, we tentatively propose a microdilution assay protocol for susceptibility testing of Paracoccidioides spp to antifungal drugs. This method may be clinically useful to predict resistance, even though further studies are needed.
Almeida-Paes, Rodrigo; Oliveira, Manoel Marques Evangelista; Freitas, Dayvison Francis Saraiva; Valle, Antônio Carlos Francesconi do; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely Maria
Sporotrichosis is a subacute to chronic infection caused by members of the Sporothrix schenckii complex. Itraconazole is the first choice antifungal drug for treating this infection, with terbinafine and potassium iodide as alternatives and amphotericin B used in cases of severe infections. Correlation of antifungal susceptibility data with the clinical outcome of the patients is scarce. The aim of this study was to correlate clinical and mycological data in patients with refractory sporotrichosis. In this work, antifungal susceptibilities, determined according to the reference M38-A2 CLSI protocol, of 25 Sporothrix strains, isolated from seven human cases of sporotrichosis with adversities in the treatment, are presented. Tested drugs included itraconazole, ketoconazole, posaconazole, voriconazole, terbinafine, and amphotericin B. Fungi were identified using the T3B PCR fingerprinting. This method identified all strains as Sporothrix brasiliensis and also demonstrated a high degree of similarity between the strains. In general, voriconazole was ineffective against all strains, and elevated minimal inhibitory concentrations (MICs) were observed for amphotericin B. High itraconazole and terbinafine MICs were not observed in S. brasiliensis isolates from patients of this study. Moreover, a significant increase in itraconazole and terbinafine MIC values from strains isolated from the same patient in different periods was not observed. The results suggest that the antifungal susceptibility to terbinafine and itraconazole determined by the reference method does not play an important role in therapeutic failure of sporotrichosis and that acquisition of resistance during prolonged antifungal treatment is not likely to occur in S. brasiliensis.
Bailão, Elisa Flávia L. C.; Lima, Patrícia de Sousa; Silva-Bailão, Mirelle G.; Bailão, Alexandre M.; Fernandes, Gabriel da Rocha; Kosman, Daniel J.; Soares, Célia Maria de Almeida
Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation. PMID:26441843
González-González, A E; Aliouat-Denis, C M; Carreto-Binaghi, L E; Ramírez, J A; Rodríguez-Arellanes, G; Demanche, C; Chabé, M; Aliouat, E M; Dei-Cas, E; Taylor, M L
Histoplasma capsulatum was sampled in lungs from 87 migratory Tadarida brasiliensis bats captured in Mexico (n=66) and Argentina (n=21). The fungus was screened by nested-PCR using a sensitive and specific Hcp100 gene fragment. This molecular marker was detected in 81·6% [95% confidence interval (CI) 73·4-89·7] of all bats, representing 71 amplified bat lung DNA samples. Data showed a T. brasiliensis infection rate of 78·8% (95% CI 68·9-88·7) in bats captured in Mexico and of 90·4% (95% CI 75·2-100) in those captured in Argentina. Similarity with the H. capsulatum sequence of a reference strain (G-217B) was observed in 71 Hcp100 sequences, which supports the fungal findings. Based on the neighbour-joining and maximum parsimony Hcp100 sequence analyses, a high level of similarity was found in most Mexican and all Argentinean bat lung samples. Despite the fact that 81·6% of the infections were molecularly evidenced, only three H. capsulatum isolates were cultured from all samples tested, suggesting a low fungal burden in lung tissues that did not favour fungal isolation. This study also highlighted the importance of using different tools for the understanding of histoplasmosis epidemiology, since it supports the presence of H. capsulatum in T. brasiliensis migratory bats from Mexico and Argentina, thus contributing new evidence to the knowledge of the environmental distribution of this fungus in the Americas.
Gonzalez-Carrillo, Carolina; Millan-Sauceda, Cassandra; Lozano-Garza, Hector Gerardo; Ortiz-Lopez, Rocio; Elizondo-Gonzalez, Ramiro; Welsh, Oliverio; Ocampo-Candiani, Jorge
Nocardia species, particularly Nocardia brasiliensis, are etiologic agents of mycetoma, a chronic subcutaneous infection. Until now, little has been known about the pathogenic mechanisms involved in nocardial infection. Traditionally, subculture in rich media has been a simple way to induce attenuation. In this work, we report the changes in virulence toward mice and in genomic constitution of N. brasiliensis produced after 200 continuous subcultures in brain heart infusion (BHI) medium (P-200 strain). The ability of the N. brasiliensis P-200 strain to produce experimental infection was tested using BALB/c mice. P-200 was also used to immunize mice to determine whether it could induce resistance against a challenge with a nonsubcultured isolate (P-0). Comparative proteomic analysis between N. brasiliensis P-0 and P-200 was performed by two-dimensional (2-D) electrophoresis, and the genome sequence was obtained through Roche 454 sequence analysis. Virulence in BALB/c mice was completely lost, and BALB/c mice immunized with P-200 bacterial cells were resistant to mycetoma production by the nonsubcultured strain. Whole-genome sequence analysis revealed that P-200 lost a total of 262,913 bp distributed in 19 deleted regions, involving a total of 213 open reading frames (ORFs). The deleted genes included those encoding bacterial virulence factors, e.g., catalase, nitrate reductase enzymes, and a group of mammalian cell entry (MCE) family proteins, which may explain the loss of virulence of the isolate. Thus, completely attenuated N. brasiliensis was obtained after 200 passages in BHI medium, and putative Nocardia virulence genes were identified for the first time. PMID:27354446
Ferreira, A C; Rosenthal, D; Carvalho, D P
Flavonoids are known inhibitors of thyroid peroxidase (TPO) and some are components of Kalanchoe brasiliensis, a plant used in popular medicine to treat tissue injuries, enlarged ganglia and peptic ulcer. As K. brasiliensis extract is currently used, the present study was designed to evaluate the effects of K. brasiliensis aqueous extract on TPO activity. We show here that TPO iodide-oxidation activity was significantly inhibited by K. brasiliensis aqueous extract and that TPO inhibition seems to be competitive, since the enzyme V(max) was unchanged and K(m) for iodide was significantly increased in the presence of the plant extract. Furthermore, TPO inhibitory activity produced by K. brasiliensis extract was unchanged after boiling or by incubation with hepatic enzymes (activated S9 fraction), suggesting that at least the antithyroid component of this plant infusion could probably reach systemic circulation. We also report that K. brasiliensis aqueous extract is able to scavenge H(2)O(2), in vitro. As H(2)O(2) is an essential TPO cofactor, it is possible that the H(2)O(2) trapping effect of K. brasiliensis may be responsible, at least in part, for the inhibition of the iodide-oxidation reaction catalysed by this enzyme. Thus, K. brasiliensis aqueous extract has antithyroid effects in vitro, suggesting that its chronic consumption could contribute to the development of goitre and hypothyroidism, mainly in areas of low iodine intake.
Lee, Dong Hwan; Kim, Jin-Beom; Lim, Jeong-A; Han, Sang-Wook; Heu, Sunggi
The plant pathogenic bacterial genus Pectobacteirum consists of heterogeneous strains. The P. carotovorum species is a complex strain showing divergent characteristics, and a new subspecies named P. carotovorum subsp. brasiliensis has been identified recently. In this paper, we re-identified the P. carotovorum subsp. brasiliensis isolates from those classified under the subspecies carotovorum and newly isolated P. carotovorum subsp. brasiliensis strains. All isolates were able to produce plant cell-wall degrading enzymes such as pectate lyase, polygalacturonase, cellulase and protease. We used genetic and biochemical methods to examine the diversity of P. carotovorum subsp. brasiliensis isolates, and found genetic diversity within the brasiliensis subsp. isolates in Korea. The restriction fragment length polymorphism analysis based on the recA gene revealed a unique pattern for the brasiliensis subspecies. The Korean brasiliensis subsp. isolates were divided into four clades based on pulsed-field gel electrophoresis. However, correlations between clades and isolated hosts or year could not be found, suggesting that diverse brasiliensis subsp. isolates existed. PMID:25288994
Bezerra, Claudia Mendonça; Cavalcanti, Luciano Pamplona de Góes; de Souza, Rita de Cássia Moreira; Barbosa, Silvia Ermelinda; Xavier, Samanta Cristina das Chagas; Jansen, Ana Maria; Ramalho, Relrison Dias; Diotaiut, Liléia
The role played by different mammal species in the maintenance of Trypanosoma cruzi is not constant and varies in time and place. This study aimed to characterise the importance of domestic, wild and peridomestic hosts in the transmission of T. cruzi in Tauá, state of Ceará, Caatinga area, Brazil, with an emphasis on those environments colonised by Triatoma brasiliensis. Direct parasitological examinations were performed on insects and mammals, serologic tests were performed on household and outdoor mammals and multiplex polymerase chain reaction was used on wild mammals. Cytochrome b was used as a food source for wild insects. The serum prevalence in dogs was 38% (20/53), while in pigs it was 6% (2/34). The percentages of the most abundantly infected wild animals were as follows: Thrichomys laurentius 74% (83/112) and Kerodon rupestris 10% (11/112). Of the 749 triatomines collected in the household research, 49.3% (369/749) were positive for T. brasiliensis, while 6.8% were infected with T. cruzi (25/369). In captured animals, T. brasiliensis shares a natural environment with T. laurentius, K. rupestris, Didelphis albiventris, Monodelphis domestica, Galea spixii, Wiedomys pyrrhorhinos, Conepatus semistriatus and Mus musculus. In animals identified via their food source, T. brasiliensis shares a natural environment with G. spixii, K. rupestris, Capra hircus, Gallus gallus, Tropidurus oreadicus and Tupinambis merianae. The high prevalence of T. cruzi in household and peridomiciliar animals reinforces the narrow relationship between the enzootic cycle and humans in environments with T. brasiliensis and characterises it as ubiquitous. PMID:25410992
Bezerra, Claudia Mendonça; Cavalcanti, Luciano Pamplona de Góes; Souza, Rita de Cássia Moreira de; Barbosa, Silvia Ermelinda; Xavier, Samanta Cristina das Chagas; Jansen, Ana Maria; Ramalho, Relrison Dias; Diotaiut, Liléia
The role played by different mammal species in the maintenance of Trypanosoma cruzi is not constant and varies in time and place. This study aimed to characterise the importance of domestic, wild and peridomestic hosts in the transmission of T. cruzi in Tauá, state of Ceará, Caatinga area, Brazil, with an emphasis on those environments colonised by Triatoma brasiliensis. Direct parasitological examinations were performed on insects and mammals, serologic tests were performed on household and outdoor mammals and multiplex polymerase chain reaction was used on wild mammals. Cytochrome b was used as a food source for wild insects. The serum prevalence in dogs was 38% (20/53), while in pigs it was 6% (2/34). The percentages of the most abundantly infected wild animals were as follows: Thrichomys laurentius 74% (83/112) and Kerodon rupestris 10% (11/112). Of the 749 triatomines collected in the household research, 49.3% (369/749) were positive for T. brasiliensis, while 6.8% were infected with T. cruzi (25/369). In captured animals, T. brasiliensis shares a natural environment with T. laurentius, K. rupestris, Didelphis albiventris, Monodelphis domestica, Galea spixii, Wiedomys pyrrhorhinos, Conepatus semistriatus and Mus musculus. In animals identified via their food source, T. brasiliensis shares a natural environment with G. spixii, K. rupestris, Capra hircus, Gallus gallus, Tropidurus oreadicus and Tupinambis merianae. The high prevalence of T. cruzi in household and peridomiciliar animals reinforces the narrow relationship between the enzootic cycle and humans in environments with T. brasiliensis and characterises it as ubiquitous.
Bezerra, Claudia Mendonça; Cavalcanti, Luciano Pamplona de Góes; Souza, Rita de Cássia Moreira de; Barbosa, Silvia Ermelinda; Xavier, Samanta Cristina das Chagas; Jansen, Ana Maria; Ramalho, Relrison Dias; Diotaiut, Liléia
The role played by different mammal species in the maintenance of Trypanosoma cruzi is not constant and varies in time and place. This study aimed to characterise the importance of domestic, wild and peridomestic hosts in the transmission of T. cruzi in Tauá, state of Ceará, Caatinga area, Brazil, with an emphasis on those environments colonised by Triatoma brasiliensis. Direct parasitological examinations were performed on insects and mammals, serologic tests were performed on household and outdoor mammals and multiplex polymerase chain reaction was used on wild mammals. Cytochrome b was used as a food source for wild insects. The serum prevalence in dogs was 38% (20/53), while in pigs it was 6% (2/34). The percentages of the most abundantly infected wild animals were as follows: Thrichomys laurentius 74% (83/112) and Kerodon rupestris 10% (11/112). Of the 749 triatomines collected in the household research, 49.3% (369/749) were positive for T. brasiliensis, while 6.8% were infected with T. cruzi (25/369). In captured animals, T. brasiliensis shares a natural environment with T. laurentius, K. rupestris, Didelphis albiventris, Monodelphis domestica, Galea spixii, Wiedomys pyrrhorhinos, Conepatus semistriatus and Mus musculus. In animals identified via their food source, T. brasiliensis shares a natural environment with G. spixii, K. rupestris, Capra hircus, Gallus gallus, Tropidurus oreadicus and Tupinambis merianae. The high prevalence of T. cruzi in household and peridomiciliar animals reinforces the narrow relationship between the enzootic cycle and humans in environments with T. brasiliensis and characterises it as ubiquitous.
Vera-Cabrera, L; Salinas-Carmona, M C; Welsh, O; Rodriguez, M A
Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed. Images PMID:1583118
González-Martínez, Norma Alejandra; Lozano-Garza, Hector Gerardo; Castro-Garza, Jorge; De Osio-Cortez, Alexandra; Vargas-Villarreal, Javier; Cavazos-Rocha, Norma; Ocampo-Candiani, Jorge; Makarov, Vadim; Cole, Stewart T.; Vera-Cabrera, Lucio
Background Mycetoma is a neglected, chronic, and deforming infectious disease caused by fungi and actinomycetes. In Mexico, N. brasiliensis is the predominant etiologic agent. Therapeutic alternatives are necessary because the current drug regimens have several disadvantages. Benzothiazinones (BTZ) are a new class of candidate drugs that inhibit decaprenyl-phosphoribose-epimerase (DprE1), an essential enzyme involved in the cell wall biosynthesis of Corynebacterineae. Methodology/Principal findings In this study, the in vitro activity of the next generation BTZ, PBTZ169, was tested against thirty Nocardia brasiliensis isolates. The MIC50 and MIC90 values for PBTZ169 were 0.0075 and 0.03 μg/mL, respectively. Because Nocardia is a potential intracellular bacterium, a THP-1 macrophage monolayer was infected with N. brasiliensis HUJEG-1 and then treated with PBTZ169, resulting in a decrease in the number of colony-forming units (CFUs) at a concentration of 0.25X the in vitro value. The in vivo activity was evaluated after infecting female BALB/c mice in the right hind food-pad. After 6 weeks, treatment was initiated with PBTZ169 and its activity was compared with the first generation compound, BTZ043. Both BTZ compounds were administered at 100 mg/kg twice daily by gavage, and sulfamethoxazole/trimethoprim (SXT), at 100 mg/kg sulfamethoxazole, was used as a positive control. After 22 weeks of therapy, only PBTZ169 and SXT displayed statistically significant activity. Conclusion These results indicate that DprE1 inhibitors may be useful for treating infections of Nocardia and may therefore be active against other actinomycetoma agents. We must test combinations of these compounds with other antimicrobial agents, such as linezolid, tedizolid or SXT, that have good to excellent in vivo activity, as well as new DprE1 inhibitors that can achieve higher plasma levels. PMID:26474057
Castro, Aline Pereira; de Mattos, Ana Carolina Alves; Pereira, Neusa Araújo; Anchieta, Naira Ferreira; Silva, Matheus Siqueira; Dias, Danielle Ferreira; Silva, Claudinei Alves; Barros, Giulliano Vilela; Souza, Raquel Lopes Martins; Dos Santos, Marcelo Henrique; Marques, Marcos José
Praziquantel is the drug of choice for the treatment of schistosomiasis. However, several strains of Schistosoma mansoni are resistant to praziquantel, making it necessary to discover new drugs that might be used for its treatment. With this in mind, the properties of a schistosomicidal ethanolic extract of Garcinia brasiliensis Mart. epicarp, the fractions obtained by partitioning this extract, including the hexane fractions, ethyl acetate fraction, and the aqueous fraction, and the isolated compounds 7-epiclusianone, a major component from these fractions, and fukugetin were tested in vitro on adult worms of S. mansoni. Mortality, damage to membranes, and excretory system activity were observed at 100.0, 50.0, 75.0, and 14.0 µg/mL for the ethanolic extract of G. brasiliensis Mart. epicarp, its hexane fractions, the ethyl acetate fraction, and 7-epiclusianone, respectively. For 7-epiclusianone, these data were confirmed by fluorescent probe Hoechst 33 258 and resorufin. Additionally, the biocidal effect of 7-epiclusianone was even higher than the hexane fractions. Moreover, an inhibitory effect of 7-epiclusianone on the egg laying of female adult S. mansoni worms was observed in cercariae and schistossomula. Thus, 7-epiclusianone is a promising schistosomicidal compound; however, more studies are needed to elucidate its mechanism of toxicity and to evaluate the in vivo activity of this compound.
Tristão, Gabriel B.; Assunção, Leandro do Prado; dos Santos, Luiz Paulo A.; Borges, Clayton L.; Silva-Bailão, Mirelle Garcia; Soares, Célia M. de Almeida; Cavallaro, Gabriele; Bailão, Alexandre M.
Approximately one-third of all proteins have been estimated to contain at least one metal cofactor, and these proteins are referred to as metalloproteins. These represent one of the most diverse classes of proteins, containing metal ions that bind to specific sites to perform catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Its function and the type of metal binder can also be predicted via a bioinformatics approach. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobic mycelia in the environment and as yeast, the parasitic form, in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. Many metalloproteins are important for the virulence of several pathogenic microorganisms. Accordingly, the present work aimed to predict the copper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides (Pb01, Pb03, and Pb18). The metalloproteins were identified using bioinformatics approaches based on structure, annotation and domains. Cu-, Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by Paracoccidioides spp. genomes. Zinc proteins were the most abundant metalloproteins, representing 5.7% of the fungus proteome, whereas copper and iron proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore, it was observed that many of these metalloproteins serve as virulence factors in the biology of the fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the Paracoccidioides spp. are of the utmost importance for the biology and virulence of these particular human pathogens. PMID:25620964
Marcos, Caroline M.; de Oliveira, Haroldo C.; da Silva, Julhiany de F.; Assato, Patrícia A.; Fusco-Almeida, Ana M.; Mendes-Giannini, Maria J. S.
Paracoccidioides species are dimorphic fungi and are the etiologic agents of paracoccidioidomycosis, which is a serious disease that involves multiple organs. The many tissues colonized by this fungus suggest a variety of surface molecules involved in adhesion. A surprising finding is that most enzymes in the glycolytic pathway, tricarboxylic acid (TCA) cycle and glyoxylate cycle in Paracoccidioides spp. have adhesive properties that aid in interacting with the host extracellular matrix and thus act as ‘moonlighting’ proteins. Moonlighting proteins have multiple functions, which adds a dimension to cellular complexity and benefit cells in several ways. This phenomenon occurs in both eukaryotes and prokaryotes. For example, moonlighting proteins from the glycolytic pathway or TCA cycle can play a role in bacterial pathogenesis by either acting as proteins secreted in a conventional pathway and/or as cell surface components that facilitate adhesion or adherence. This review outlines the multifunctionality exhibited by many Paracoccidioides spp. enzymes, including aconitase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate lyase, malate synthase, triose phosphate isomerase, fumarase, and enolase. We discuss the roles that moonlighting activities play in the virulence characteristics of this fungus and several other human pathogens during their interactions with the host. PMID:25566229
Clavijo-Giraldo, Diana M; Matínez-Alvarez, José A; Lopes-Bezerra, Leila M; Ponce-Noyola, Patricia; Franco, Bernardo; Almeida, Ricardo S; Mora-Montes, Héctor M
The study of the host-pathogen interaction is essential to understand the mechanisms underlying adhesion, colonization and tissue damage by pathogens. This is usually achieved by performing in vivo studies using small mammals, such as rats, mice and guinea pigs. Nowadays, the mouse models of systemic or subcutaneous infection are the gold standard assays to analyze the virulence of members of the Sporothrix schenckii complex. There are, however, invertebrates that have been recently used as alternative hosts to assess the virulence of both bacteria and fungi, and among them, larvae of Galleria mellonella are popular because they are easy to breed, and require non-specialized facilities to maintain the colony. Here, we assessed the use of G. mellonella larvae to test the virulence of S. schenckii sensu stricto and Sporothrix brasiliensis strains, and found that infection with yeast-like cells, but not with conidia or germlings, reproduces the virulence data generated in the mouse model of infection. Furthermore, with this insect model we could classify the virulence of some strains as low, intermediate or high, in line with the observations in the mammalian model. Therefore, G. mellonella is suitable, and a new alternative, to test virulence of both S. schenckii sensu stricto and S. brasiliensis.
Hurtado Páez, Uriel Alonso; García Romero, Ibonne Aydee; Restrepo Restrepo, Silvia; Aristizábal Gutiérrez, Fabio Ancizar; Montoya Castaño, Dolly
Natural rubber (Hevea brasiliensis) is a tropical tree used commercially for the production of latex, from which 40,000 products are generated. The fungus Microcyclus ulei infects this tree, causing South American leaf blight (SALB) disease. This disease causes developmental delays and significant crop losses, thereby decreasing the production of latex. Currently several groups are working on obtaining clones of rubber tree with durable resistance to SALB through the use of extensive molecular biology techniques. In this study, we used a secondary clone that was resistant to M. ulei isolate GCL012. This clone, FX 3864 was obtained by crossing between clones PB 86 and B 38 (H. brasiliensis x H. brasiliensis). RNA-Seq high-throughput sequencing technology was used to analyze the differential expression of the FX 3864 clone transcriptome at 0 and 48 h post infection (hpi) with the M. ulei isolate GCL012. A total of 158,134,220 reads were assembled using the de novo assembly strategy to generate 90,775 contigs with an N50 of 1672. Using a reference-based assembly, 76,278 contigs were generated with an N50 of 1324. We identified 86 differentially expressed genes associated with the defense response of FX 3864 to GCL012. Seven putative genes members of the AP2/ERF ethylene (ET)-dependent superfamily were found to be down-regulated. An increase in salicylic acid (SA) was associated with the up-regulation of three genes involved in cell wall synthesis and remodeling, as well as in the down-regulation of the putative gene CPR5. The defense response of FX 3864 against the GCL012 isolate was associated with the antagonistic SA, ET and jasmonic acid (JA) pathways. These responses are characteristic of plant resistance to biotrophic pathogens.
Restrepo Restrepo, Silvia; Aristizábal Gutiérrez, Fabio Ancizar; Montoya Castaño, Dolly
Natural rubber (Hevea brasiliensis) is a tropical tree used commercially for the production of latex, from which 40,000 products are generated. The fungus Microcyclus ulei infects this tree, causing South American leaf blight (SALB) disease. This disease causes developmental delays and significant crop losses, thereby decreasing the production of latex. Currently several groups are working on obtaining clones of rubber tree with durable resistance to SALB through the use of extensive molecular biology techniques. In this study, we used a secondary clone that was resistant to M. ulei isolate GCL012. This clone, FX 3864 was obtained by crossing between clones PB 86 and B 38 (H. brasiliensis x H. brasiliensis). RNA-Seq high-throughput sequencing technology was used to analyze the differential expression of the FX 3864 clone transcriptome at 0 and 48 h post infection (hpi) with the M. ulei isolate GCL012. A total of 158,134,220 reads were assembled using the de novo assembly strategy to generate 90,775 contigs with an N50 of 1672. Using a reference-based assembly, 76,278 contigs were generated with an N50 of 1324. We identified 86 differentially expressed genes associated with the defense response of FX 3864 to GCL012. Seven putative genes members of the AP2/ERF ethylene (ET)-dependent superfamily were found to be down-regulated. An increase in salicylic acid (SA) was associated with the up-regulation of three genes involved in cell wall synthesis and remodeling, as well as in the down-regulation of the putative gene CPR5. The defense response of FX 3864 against the GCL012 isolate was associated with the antagonistic SA, ET and jasmonic acid (JA) pathways. These responses are characteristic of plant resistance to biotrophic pathogens. PMID:26287380
Colauto, Nelson Barros; da Silveira, Adriano Reis; da Eira, Augusto Ferreira; Linde, Giani Andrea
Casing layer is one of the most important components of Agaricus spp. production and it directly affects mushroom productivity, size and mass. The aim of this study was to evaluate potential raw materials as a casing layer and their effect on Agaricus brasiliensis productivity. Raw materials from Brazil with potential use were selected and characterized, and the most promising ones were tested as casing layers for mushroom yield. Evaluated raw materials included lime schist, vermiculite, eucalyptus sawdust, sand, São Paulo peat, Santa Catarina peat, subsoil and charcoal. Particle size, porosity and water absorption in relation to mushroom yield for casing layers were determined. Lime schist, an alternate casing layer to peat, is presented and the effects of the casing layer on the mushroom yield are discussed.
Lorenzo, M G; Guarneri, A A; Pires, H H; Diotaiuti, L; Lazzari, C R
Vector-borne transmission of Chagas disease in Northeast Brazil is basically by Triatoma brasiliensis. It is thus crucial to determine this species' microclimatic preferences as limiting factors for its distribution and ability to infest domestic environments. We analyze the microclimatic properties of the shelters in which these insects are found in wild, domestic, and peridomiciliary environments in the State of Ceará, at Brazil. We measure temperature and relative humidity (RH) every 15 minutes for 3 days. Thermal variation was greatly dampened inside both domiciliary refuges and the more protected internal places in wild stony sites. For RH, we observed a similar dampening pattern, but mean RH was lower in both domiciliary refuges and wild ones inside stony sites as compared to reference levels in the surrounding environment. The results are discussed with regard to this species' microclimatic preferences in the laboratory and its potential as determinants of its geographical distribution.
Araújo, Felipe Souto; Coelho, Luciene Melo; Silva, Lívia do Carmo; da Silva Neto, Benedito Rodrigues; Parente-Rocha, Juliana Alves; Bailão, Alexandre Melo; de Oliveira, Cecília Maria Alves; Fernandes, Gabriel da Rocha; Hernández, Orville; Ochoa, Juan Guillermo McEwen; Soares, Célia Maria de Almeida; Pereira, Maristela
Paracoccidioides spp., a dimorphic pathogenic fungus, is the etiologic agent of paracoccidioidomycosis (PCM). PCM is an endemic disease that affects at least 10 million people in Latin America, causing severe public health problems. The drugs used against pathogenic fungi have various side effects and limited efficacy; therefore, there is an inevitable and urgent medical need for the development of new antifungal drugs. In the present study, we evaluated the transcriptional profile of Paracoccidioides lutzii exposed to argentilactone, a constituent of the essential oil of Hyptis ovalifolia. A total of 1,058 genes were identified, of which 208 were up-regulated and 850 were down-regulated. Cell rescue, defense and virulence, with a total of 26 genes, was a functional category with a large number of genes induced, including heat shock protein 90 (hsp90), cytochrome c peroxidase (ccp), the hemoglobin ligand RBT5 (rbt5) and superoxide dismutase (sod). Quantitative real-time PCR revealed an increase in the expression level of all of those genes. An enzymatic assay showed a significant increase in SOD activity. The reduced growth of Pbhsp90-aRNA, Pbccp-aRNA, Pbsod-aRNA and Pbrbt5-aRNA isolates in the presence of argentilactone indicates the importance of these genes in the response of Paracoccidioides spp. to argentilactone. The response of the P. lutzii cell wall to argentilactone treatment was also evaluated. The results showed that argentilactone caused a decrease in the levels of polymers in the cell wall. These results suggest that argentilactone is a potential candidate for antifungal therapy. PMID:26734764
Oliveira, Isabel Araújo; Argolo, Leandro Araújo; Bitencourt, Jamille de Araújo; Diniz, Débora; Vicari, Marcelo Ricardo; Affonso, Paulo Roberto Antunes de Mello
Among American cichlids, Geophagus stands out as a species-rich genus widespread over neotropical region. Despite their diversity and confusing taxonomy, only few and basic chromosomal reports are available in populations/species along Atlantic coast, hindering our understanding about evolutionary trends in this genus. Therefore, detailed chromosomal studies were performed in "Geophagus" brasiliensis complex from coastal rivers in northeastern Brazil, totalizing seven populations of Geophagus brasiliensis and one of Geophagus itapicuruensis. All samples shared a diploid number (2n) of 48 divided into 2 submetacentric and 46 subtelocentric/acrocentric chromosomes with (peri)centromeric heterochromatin, hypothesized as a symplesiomorphy for Geophagini. Although G. itapicuruensis and two populations of G. brasiliensis presented single NORs on short arms, multiple GC-rich rDNA sites were observed in the remaining G. brasiliensis samples, ranging from three to six NOR-bearing chromosomes. Inversely, 5S rDNA sites were invariably located at interstitial region on a st/a pair, nonsyntenic to NORs. A compilation of the data set in Geophagus shows that their chromosomal evolution has been driven by pericentric inversions and microstructural changes. Besides, the divergence found in northeastern Brazil places this region as a biodiversity hotspot. A taxonomic revision in the complex "Geophagus" brasiliensis is recommended with the support of cytogenetic analyses.
Background The Triatoma brasiliensis complex is a monophyletic group, comprising three species, one of which includes two subspecific taxa, distributed across 12 Brazilian states, in the caatinga and cerrado biomes. Members of the complex are diverse in terms of epidemiological importance, morphology, biology, ecology, and genetics. Triatoma b. brasiliensis is the most disease-relevant member of the complex in terms of epidemiology, extensive distribution, broad feeding preferences, broad ecological distribution, and high rates of infection with Trypanosoma cruzi; consequently, it is considered the principal vector of Chagas disease in northeastern Brazil. Methods We used ecological niche models to estimate potential distributions of all members of the complex, and evaluated the potential for suitable adjacent areas to be colonized; we also present first evaluations of potential for climate change-mediated distributional shifts. Models were developed using the GARP and Maxent algorithms. Results Models for three members of the complex (T. b. brasiliensis, N = 332; T. b. macromelasoma, N = 35; and T. juazeirensis, N = 78) had significant distributional predictivity; however, models for T. sherlocki and T. melanica, both with very small sample sizes (N = 7), did not yield predictions that performed better than random. Model projections onto future-climate scenarios indicated little broad-scale potential for change in the potential distribution of the complex through 2050. Conclusions This study suggests that T. b. brasiliensis is the member of the complex with the greatest distributional potential to colonize new areas: overall; however, the distribution of the complex appears relatively stable. These analyses offer key information to guide proactive monitoring and remediation activities to reduce risk of Chagas disease transmission. PMID:24886587
Peres, Rafael; Amaral, Fernanda Gaspardo; Marques, Antonio Carlos; Neto, José Cipolla
The primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the tree of life, in animals, plants, and fungi, supporting a deep evolutionary origin for this signaling molecule. However, some key groups have not been studied. Echinoderms, deuterostome animals, are one of these groups. Herein we study the presence of melatonin and enzymes of its pathway in the sea star Echinaster brasiliensis. We demonstrate that E. brasiliensis produces endogenous melatonin, in the gonads, under a circadian pattern with a nocturnal peak of production. We also show that the enzymes arylalkylamine N-acetyltransferase (AANAT) and tryptophan hydroxylase (TPH) are present and are probably regulating the melatonin production.
Neiss, Ulisses Gaspar; Hamada, Neusa
The larva of Palaemnema brasiliensis Machado, 2009 is described and illustrated based on last-instar larvae and exuviae of reared larvae collected in a blackwater stream in Barcelos and Presidente Figueiredo municipalities, Amazonas state, Brazil. The larva of P. brasiliensis can be distinguished from the two South American species of the genus with described larvae (P. clementia Selys and P. mutans Calvert), mainly by presence of a single obtuse cusp on the labial palp, the presence and configuration of setae in the caudal lamellae, and the proportional length of terminal filaments of the caudal lamellae. The family is recorded here for the first time in Brazilian state of Amazonas.
de Arruda Grossklaus, Daciene; Bailão, Alexandre Melo; Vieira Rezende, Tereza Cristina; Borges, Clayton Luiz; de Oliveira, Milton Adriano Pelli; Parente, Juliana Alves; de Almeida Soares, Célia Maria
An efficient oxidative stress response is important to the fungal pathogen Paracoccidioides to survive within the human host. In this study, oxidative stress was mimicked by exposure of yeast cells to hydrogen peroxide (2 mM H2O2). To investigate the effect of H2O2 on the proteome of Paracoccidioides, we used a large scale 2-DE protein gel electrophoresis approach to analyze differentially expressed proteins/isoforms that were detected in early (2 h) and in late (6 h) oxidative stress treatments. All proteins/isoforms were grouped based on their functional categories that revealed a global activation of antioxidant enzymes, such as catalase, superoxide dismutase, cytochrome C peroxidase and thioredoxin. A view of the metabolic cell profile, as determined by proteomics, depicted a shift in the yeast cells metabolism as suggested by the activation of the pentose phosphate pathway, a great source of cellular reducing power in the form of NADPH. Additionally, in silico analyzes depicted 34 oxidoreductases proteins/isoforms putatively involved with defense against oxidative stress. Confirmatory assays of enzymatic activity, flow cytometry, transcript levels and NADPH measurements, produced data in agreement with proteomic analysis.
Oliveira, Jader; Marcet, Paula L; Takiya, Daniela M; Mendonça, Vagner J; Belintani, Tiago; Bargues, Maria D; Mateo, Lucia; Chagas, Vivian; Folly-Ramos, Elaine; Cordeiro-Estrela, Pedro; Gurgel-Gonçalves, Rodrigo; Costa, Jane; da Rosa, João A; Almeida, Carlos E
"Triatoma brasiliensis species complex" was defined as a monophyletic group of the species: T. brasiliensis, T. juazeirensis, T. melanica, and T. sherlocki. An alternative grouping scheme proposed the concept of "Brasiliensis subcomplex" which included the former species together with T. melanocephala, T. petrocchiae, T. lenti, T. tibiamaculata, and T. vitticeps. To evaluate the relationship among these taxa we combined the results obtained with four mitochondrial genes (12S, 16S, COI and Cytb, adding to 1811bp) and geometric morphometric analysis of wings and heads. Panstrongylus megistus was included in the analysis as it was previously found related to T. tibiamaculata, T. melanocephala and T. vitticeps. The results of both molecular and morphometric approaches clearly grouped the species analyzed into two monophyletic units, supported by both genetic and wing variability. The first one (G1) comprises the four species originally included in the T. brasiliensis species complex plus T. lenti and T. petrocchiae. The second group (G2) was composed by T. melanocephala, T. tibiamaculata and T. vitticeps, and remarkably, P. megistus if considering wing variability and phylogenetic results. Nevertheless, geometric morphometrics of heads provided a quantitative measurement that discriminates Panstrongylus from the Triatoma species based on the position of the antennal insertion relative to eyes, as it is used as the generic distinctive character. The discrepancy among approaches questions the validity of this character to define Panstrongylus genus. Independently of the chosen group definition -"T. brasiliensis species complex" or "Brasiliensis subcomplex"-we propose to delimit it to species of G1 that are all associated with the Caatinga biome in the Brazilian Northeast. G2 are the ones associated with the Atlantic Forest biome.
Turmelle, Amy S; Allen, Louise C; Schmidt-French, Barbara A; Jackson, Felix R; Kunz, Thomas H; McCracken, Gary F; Rupprecht, Charles E
A captive colony of Brazilian free-tailed bats (Tadarida brasiliensis) was vaccinated with a commercial monovalent inactivated rabies virus (RABV) vaccine (RABVAC 1). Baseline rabies virus neutralizing antibodies (VNA) and the response to vaccination were measured in 50 bats. Rabies VNA was detected in the plasma of 64% (27/42) of bats that had been vaccinated 1 yr prior, but only 19% (8/42) had levels considered adequate. Rabies VNA was detected in the plasma of 63% (5/8) of bats with no record of previous vaccination, suggesting natural RABV exposure before captivity. All bats demonstrated a VNA response by 10 days postvaccination, and baseline titer significantly predicted humoral response to vaccination. No adverse reactions to vaccination or clinical signs of RABV infection were observed in the bats during a 6-mo observation period. Annual vaccination may maintain immunity against RABV infection in captive colonies of bats. Bat, rabies virus, Tadarida brasiliensis, vaccination, virus neutralizing antibodies.
Nelson, Shakira M.; Shay, Ashley E.; James, Jamaal L.; Carlson, Bradley A.; Urban, Joseph F.; Prabhu, K. Sandeep
The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) toward an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary selenium modulates macrophage polarization toward an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a selenium-adequate (0.08 ppm) diet significantly augmented intestinal AAM presence while decreasing adult worms and fecal egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diet. Further increase in dietary selenium to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the selenium-supplemented Trspfl/flCreWT mice that express selenoproteins driven by tRNASec (Trsp), whereas N. brasiliensis-infected Trspfl/flCreLysM selenium-supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in selenium-adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of selenium supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARγ blocked the effect of selenium. These results suggest that optimal expression of selenoproteins and selenium-dependent production of COX-derived endogenous prostanoids, such as Δ12-PGJ2 and 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses. PMID:26644468
Bravo, Eduar A; Zegarra, Arturo J; Piscoya, Alejandro; Pinto, José L; de Los Rios, Raúl E; Prochazka, Ricardo A; Huerta-Mercado, Jorge L; Mayo, Nancy L; Tagle, Martin
South American blastomycosis is a systemic micosis caused by infection with Paracoccidioides brasiliensis. The most frequently affected sites are the lower lip buccal mucous membrane, palate, tongue, sublingual region, lymph glands, and lungs. However, colonic involvement is not a common expression of Paracoccidioidomycosis. We report a case of chronic diarrhea and pancolitis caused by Paracoccidioidomycosis with fatal outcome.
Bravo, Eduar A.; Zegarra, Arturo J.; Piscoya, Alejandro; Pinto, José L.; de los Rios, Raúl E.; Prochazka, Ricardo A.; Huerta-Mercado, Jorge L.; Mayo, Nancy L.; Tagle, Martin
South American blastomycosis is a systemic micosis caused by infection with Paracoccidioides brasiliensis. The most frequently affected sites are the lower lip buccal mucous membrane, palate, tongue, sublingual region, lymph glands, and lungs. However, colonic involvement is not a common expression of Paracoccidioidomycosis. We report a case of chronic diarrhea and pancolitis caused by Paracoccidioidomycosis with fatal outcome. PMID:20671977
de Oliveira, Mônica Rocha
The reproductive aspects of Hemiramphus brasiliensis were analyzed with a view to verify the temporal dynamics of reproduction. This paper presents data on sex ratio, length at first sexual maturity, macroscopic and histological aspects of gonad development, gonadosomatic index (GSI), reproductive period, and fecundity of H. brasiliensis. The fishes were captured from the coastal waters of Rio Grande do Norte, northeastern Brazil. Females of this species predominated in the sampled population and were larger in size than the males. The length at the first sexual maturation of males was 20.8 cm and that of females was 21.5 cm. The macroscopic characteristics of the gonads indicated four maturation stages. Histological studies of gonads of H. brasiliensis showed six phases of oocyte development and four phases of spermatocyte development. The batch fecundity of this species was 1153 (±258.22) mature oocytes for 50 g body weight of female. The microscopic characteristics of gonad development indicate that H. brasiliensis is a multiple spawner, presenting a prolonged reproductive period during the whole year, with a peak in the month of April, and is considered as an opportunistic strategist. PMID:25512946
Muñoz, Pamela; Fredes, Fernando; Raffo, Eduardo; González-Acuña, Daniel; Muñoz, Lisandro; Cid, Cesar
One hundred and ninety five specimens of free tailed bat (Tadarida brasiliensis) obtained from two regions of Chile were analized to determine parasite infection. From those specimens the endoparasites identified were: Trematoda: Acanthatrium lunatum, Limatuoides limatulus and Paralecithodendrium carlsbadensis; Cestoda: Vampirolepis sp.; Nematoda: Nochtia pilosus and Anoplostrongylus paradoxus. The ectoparasites identified were Chiroptonyssus robustipes, Ewingnana inaequalis and Notoedres lasionycteris all of them are acari species. Even thought the bat specimens are from the same species on both survey sites, the results differ for each site when the parasite species identified are compared.
Shankar, Jata; Wu, Thomas D.; Clemons, Karl V.; Monteiro, Jomar P.; Mirels, Laurence F.; Stevens, David A.
Background Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17β-estradiol, E2) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E2 inhibits M-to-Y transition. Methodology We assessed temporal gene expression in strain Pb01 in the presence or absence of E2 at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E2-regulated clones were sequenced to identify genes and biological function. Principal Findings E2-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p≤0.001). Genes with low expression after 4 or 12 h exposure to E2 belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, α-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E2-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E2, whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E2. Conclusion This study characterizes the effect of E2 at the molecular level on the
Background Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. Results Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. Conclusions The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber. PMID:23375136
Fernandes, Geisa Ferreira; dos Santos, Priscila Oliveira; Rodrigues, Anderson Messias; Sasaki, Alexandre Augusto; Burger, Eva; de Camargo, Zoilo Pires
A comparative study about protein secretion, immunogenicity and virulence was performed in order to characterize and to compare eight Sporothrix schenckii sensu stricto isolates. For virulence characterization, a murine model, based on survival assay and CFU counting was used. S. brasiliensis and S. globosa, a highly virulent and a non-virulent isolates, respectively were used as external controls. Exoantigen profiles showed different secreted molecules; the 46- and 60-kDa molecules were commonly secreted by all three species. The S. schenckii s. str. isolates could be classified as non-virulent or presenting low, medium or high virulence, based on survival times after infection and recovery of viable fungi. The humoral response profiles of mice infected with S. schenckii s. str., S. globosa and S. brasiliensis were heterogeneous; five virulent isolates (S. schenckii s. str., n = 4 and S. brasiliensis, n = 1) had in common the recognition of the 60-kDa molecule by their respective antisera, suggesting that this antigen may be involved in virulence. Furthermore, the 110-kDa molecule was secreted and recognized by antisera from four virulent isolates (S. schenckii s. str., n = 3 and S. brasiliensis, n = 1), so there is a possibility that this molecule is also related to virulence. Our findings reveal different degrees of virulence in S. schenckii s. str. isolates and suggest the correlation of protein secretion and immunogenicity with virulence of S. schenckii complex. These findings provide new insights into the pathogenesis of S. schenckii s. str. and improve the knowledge about immunogenicity and protein profiles in S. schenckii complex. PMID:23324498
Natural rubber is an important commodity industrial crop that mainly derives from Hevea brasiliensis. Most natural rubber production is in Southeast Asia, but significant cultivar development takes place in Brazil, the original origin of current commercial H. brasiliensis cultivars. Thus it is criti...
Nondillo, Aline; Sganzerla, Vânia Maria Ambrosi; Bueno, Odair Correa; Botton, Marcos
Eurhizococcus brasiliensis (Wille) (Hemiptera: Margarodidae) is a soil scale that is considered the main pest of vineyards in Brazil. The ant Linepithema micans (Forel) (Hymenoptera: Formicidae) is frequently found associated with this species of scale in infested areas. The effect of the presence of L. micans on the infestation and dispersal capacity of E. brasiliensis on vine roots was measured in a greenhouse, using Paulsen 1103 rootstock seedlings planted in simple and double "Gallotti Cages." Treatments measured were: infestation of roots with E. brasiliensis or L. micans, and infestation with both species together. In the experiment using simple Gallotti Cages, with E. brasiliensis associated with L. micans, higher mean numbers of cysts and ants per plant were recorded, a result significantly different from that found for infestation with scale only. When double Gallotti Cages were used, first-instar nymphs were transported between the cages. The results showed that L. micans transports and aids in the attachment of E. brasiliensis to vine plants.
Sarquis, Otília; Carvalho-Costa, Filipe Anibal; Toma, Helena Keiko; Georg, Ingebourg; Burgoa, Marcelo R; Lima, Marli Maria
An entomological survey was carried out in four rural localities situated in the state of Ceará, assessing Chagas disease seroprevalence in man, focusing on the presence of vectors in natural foci contiguous to the domestic and peridomestic environments. Fifty-three Triatoma brasiliensis, nine T. pseudomaculata and 71 Rhodnius nasutus were collected in their natural habitats as far as 10 m from the houses, and 663, 59 and 8 respectively were captured in peridomestic artificial structures, adjacent to the houses, including henhouses, pigpens, corrals, perches and piles of bricks, tiles and wood. Within the households, 37 T. brasiliensis, one specimen of T. pseudomaculata and one of R. nasutus were captured. Overall, Trypanosoma cruzi infection rates were 2.3% for T. brasiliensis and 11.3% for R. nasutus. Despite that the seroprevalence survey in man did not reveal positive results using two serological techniques, natural triatomine habitats are juxtaposed to man-made artificial ecotopes, resulting in overlapping habitats. The contiguity between natural ecotopes and human dwellings increases the interaction between vectors and humans, challenging continuous surveillance and vector control efforts.
Steece, R S; Calisher, C H
Fetuses were collected from four Mexican free-tailed bats (Tadarida brasiliensis mexicana) and a fetal bat cell (FBC) line was established and tested for its ability to support the replication of the ERA vaccine strain of rabies virus. Cytopathic effects were detected in ERA virus-inoculated as well as uninoculated FBC's. Immunofluorescent antibody testing of uninoculated FBC's provided no evidence for the presence of rabies virus. However, mice inoculated intracranially with supernatant fluid from uninoculated FBC's died. Enzyme-linked immunosorbent assay and immunofluorescent antibody testing revealed rabies virus in the brains of these mice. Tests with a panel of monoclonal antibodies indicated that the isolate was the same as that isolated from Mexican free-tailed bats from the southwestern United States. We conclude that the fetuses from which the FBC line was derived had been infected in utero with rabies virus. We believe this may represent the first observation of prenatal transfer of rabies virus in naturally infected bats.
standing, diagnosis, and treatment of musculoskeletal infections. Key Words: musculoskeletal infection, biofilm , bacteria, biomaterial (J Orthop Trauma...form a biofilm , or slime layer.1 The recurrence of infections is often the result of microbial biofilm formation on the implant, enabling the persistence...Klebsiella pneumoniae). Staphylococcus species is by far the most studied pathogen in musculoskeletal infections and can produce a multilayered biofilm
Costa, Jane; Correia, Nathália Cordeiro; Neiva, Vanessa Lima; Gonçalves, Teresa Cristina Monte; Felix, Márcio
Triatoma brasiliensis macromelasoma is revalidated based on the results of previous multidisciplinary studies on the Triatoma brasiliensis complex, consisting of crossing experiments and morphological, biological, ecological and molecular analyses. These taxonomic tools showed the closest relationship between T. b. macromelasoma and Triatoma brasiliensis brasiliensis. T. b. macromelasoma is redescribed based on specimens collected in the type locality and specimens from a F1 colony. The complex now comprises T. b. brasiliensis, T. b. macromelasoma, Triatoma melanica, Triatoma juazeirensis and Triatoma sherlocki. An identification key for all members of the complex is presented. This detailed comparative study of the morphological features of T. b. macromelasoma and the remaining members of the complex corroborates results from multidisciplinary analyses, suggesting that the subspecific status is applicable. This subspecies can be distinguished by the following combination of features: a pronotum with 1+1 narrow brownish-yellow stripes on the submedian carinae, not attaining its apex, hemelytra with membrane cells darkened on the central portion and legs with an incomplete brownish-yellow ring on the apical half of the femora. Because the T. brasiliensis complex is of distinct epidemiological importance throughout its geographic distribution, a precise identification of its five members is important for monitoring and controlling actions against Chagas disease transmission. PMID:24037202
Barboza-Filho, Carlos G; Cabrera, Flávio C; Dos Santos, Renivaldo J; De Saja Saez, Jose Antonio; Job, Aldo E
The development of nanotechnology has generated new means of disease diagnosis and treatment. Infectious diseases, including leishmaniasis, malaria, etc., have benefited from the advent of new nanomaterials and/or nanodevices capable of detecting specific antigens and antibodies with high specificity and low cost. In this paper, we present an investigation on a single-celled protozoan Leishmaniasis parasite, a disease considered of standard infectivity, given the high degree of immunological specificity. Natural rubber (NR) membranes incorporating gold nanoparticles (GNPs) were placed in the culture medium and the physiological behavior of Leishmania brasiliensis promastigotes was evaluated. The natural rubber membranes containing GNPs decreased the population growth rate, showing a lower index of living promastigotes (attached to the membrane surface) depending on the amount of nanoparticles deposited in the membrane surface. Such membranes may be used to develop a flexible band-aid for skin lesions from degenerative infection state, inhibiting the population growth of parasites in the lesions. In addition, natural rubber membranes would also stimulate angiogenesis in damaged tissues.
Trindade-Silva, Amaro E.; Rua, Cintia; Silva, Genivaldo G. Z.; Dutilh, Bas E.; Moreira, Ana Paula B.; Edwards, Robert A.; Hajdu, Eduardo; Lobo-Hajdu, Gisele; Vasconcelos, Ana Tereza; Berlinck, Roberto G. S.; Thompson, Fabiano L.
The endemic marine sponge Arenosclera brasiliensis (Porifera, Demospongiae, Haplosclerida) is a known source of secondary metabolites such as arenosclerins A-C. In the present study, we established the composition of the A. brasiliensis microbiome and the metabolic pathways associated with this community. We used 454 shotgun pyrosequencing to generate approximately 640,000 high-quality sponge-derived sequences (∼150 Mb). Clustering analysis including sponge, seawater and twenty-three other metagenomes derived from marine animal microbiomes shows that A. brasiliensis contains a specific microbiome. Fourteen bacterial phyla (including Proteobacteria, Cyanobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Cloroflexi) were consistently found in the A. brasiliensis metagenomes. The A. brasiliensis microbiome is enriched for Betaproteobacteria (e.g., Burkholderia) and Gammaproteobacteria (e.g., Pseudomonas and Alteromonas) compared with the surrounding planktonic microbial communities. Functional analysis based on Rapid Annotation using Subsystem Technology (RAST) indicated that the A. brasiliensis microbiome is enriched for sequences associated with membrane transport and one-carbon metabolism. In addition, there was an overrepresentation of sequences associated with aerobic and anaerobic metabolism as well as the synthesis and degradation of secondary metabolites. This study represents the first analysis of sponge-associated microbial communities via shotgun pyrosequencing, a strategy commonly applied in similar analyses in other marine invertebrate hosts, such as corals and algae. We demonstrate that A. brasiliensis has a unique microbiome that is distinct from that of the surrounding planktonic microbes and from other marine organisms, indicating a species-specific microbiome. PMID:22768320
Salinas-Carmona, Mario C.; Torres-Lopez, Ernesto; Ramos, Alma I.; Licon-Trillo, Angel; Gonzalez-Spencer, Daniel
Nine- to twelve-week-old BALB/c mice were injected in footpads with 107 CFU of a Nocardia brasiliensis cell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-γ) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-γ increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection. PMID:10225905
Hong, H; Xiao, H; Yuan, H; Zhai, J; Huang, X
Mechanical wounding or treatment with exogenous jasmonates (JA) induces differentiation of the laticifer in Hevea brasiliensis. JA is a key signal for latex biosynthesis and wounding response in the rubber tree. Identification of JAZ (jasmonate ZIM-domain) family of proteins that repress JA responses has facilitated rapid progress in understanding how this lipid-derived hormone controls gene expression and related physiological processes in plants. In this work, the full-length cDNAs of six JAZ genes were cloned from H. brasiliensis (termed HbJAZ). These HbJAZ have different lengths and sequence diversity, but all of them contain Jas and ZIM domains, and two of them contain an ERF-associated amphiphilic repression (EAR) motif in the N-terminal. Real-time RT-PCR analyses revealed that HbJAZ have different expression patterns and tissue specificity. Four HbJAZ were up-regulated, one was down-regulated, while two were less effected by rubber tapping treatment, suggesting that they might play distinct roles in the wounding response. A yeast two-hybrid assay revealed that HbJAZ proteins interact with each other to form homologous or heterogeneous dimer complexes, indicating that the HbJAZ proteins may expand their function through diverse JAZ-JAZ interactions. This work lays a foundation for identification of the JA signalling pathway and molecular mechanisms of latex biosynthesis in rubber trees.
Lima, Francisco Esmaile de Sales; Campos, Fabrício Souza; Kunert Filho, Hiran Castagnino; Batista, Helena Beatriz de Carvalho Ruthner; Carnielli, Pedro; Cibulski, Samuel Paulo; Spilki, Fernando Rosado; Roehe, Paulo Michel; Franco, Ana Cláudia
A survey was carried out in search for bat coronaviruses in an urban maternity roost of about 500 specimens of two species of insectivorous bats, Molossus molossus and Tadarida brasiliensis, in Southern Brazil. Twenty-nine out of 150 pooled fecal samples tested positive by reverse transcription-PCR contained fragments of the RNA-dependent RNA polymerase gene of coronavirus-related viruses. The sequences clustered along with bat alphacoronaviruses, forming a subcluster within this group. Our findings point to the need for risk assessment and continued surveillance of coronavirus infections of bats in Brazil.
... 23(4):251-69. Association for Professionals in Infection Control and Epidemiology (APIC) guideline. Back to Top Administration ... : Hospital Scope | Glossary | References | Site Map | Credits Freedom of ...
Pedersen, Mads; Lauritzen, Henrik Klitgaard; Frisvad, Jens Christian; Meyer, Anne S
Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable beta-xylosidases. The beta-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75 degrees C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60 degrees C. At 75 degrees C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable beta-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.
Natural rubber biosynthesis occurs in laticifers of Ficus elastica and Hevea brasiliensis, and in parenchyma cells of Parthenium argentatum. Natural rubber is synthesized by rubber transferase using allylic pyrophosphates as initiators, isopentenyl pyrophosphate as monomeric substrate and magnesium ...
Thirteen nuclear-encoded microsatellites from a genomic DNA library of Serra Spanish mackerel, Scomberomorus brasiliensis, were isolated and characterized. The microsatellites include 10 perfect repeats (8 tetranucleotide and 2 dinucleotide) and 3 imperfect repeats (2 tetranucleotide and 1 dinucleo...
Marimon, Rita; Cano, Josep; Gené, Josepa; Sutton, Deanna A.; Kawasaki, Masako; Guarro, Josep
Sporothrix schenckii is the species responsible for sporotrichosis, a fungal infection caused by the traumatic implantation of this dimorphic fungus. Recent molecular studies have demonstrated that this species constitutes a complex of numerous phylogenetic species. Since the delineation of such species could be of extreme importance from a clinical point of view, we have studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strains of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences. The molecular analysis revealed that Sporothrix albicans, S. inflata, and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phenetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, and S. mexicana. The key phenotypic features for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37°C, and the assimilation of sucrose, raffinose, and ribitol. PMID:17687013
Pardo, Martha; Bonifaz, Alexandro; Valencia, Adriana; Araiza, Javier; Mejia, Silvia Anett; Mena-Cedillos, Carlos
We describe the case of a 14-year-old girl with Down syndrome and a large cutaneous plaque localized to the right neck and shoulder that had enlarged over five years after a minor traumatic injury. The plaque was characterized by numerous inflammatory nodules and fistulae that secreted purulent discharge. Nocardia grains were identified and Nocardia brasiliensis was identified by culture. Histopathology examination showed a chronic inflammatory infiltrate with granuloma development. The treatment scheme was with Diaminodiphenylsulfone 50/mg/d and Trimethoprim-Sulfamethoxazole 800/160 mg BID. Therapy was continued over 1(1/2) years, with a tapering dose. After 2(1/2) years of continuous treatment, clinical and microbiological healing was achieved.
inapparent infection. A refeeding program may thus become complicated by the sudden appearance of a life-threatening infectious illness (3). (3) The...Beisel, W. R. 23 Unusually low serum concentrations of inorganic phosphate have been reported in patients with gram-negative sepsis and in Reye’s syndrome ...infection should be corrected by a well-managed program of convalescent-period refeeding . This aspect of nutritional support is too often ignored. On the
Desjardins, Christopher A.; Champion, Mia D.; Holder, Jason W.; Muszewska, Anna; Goldberg, Jonathan; Bailão, Alexandre M.; Brigido, Marcelo Macedo; Ferreira, Márcia Eliana da Silva; Garcia, Ana Maria; Grynberg, Marcin; Gujja, Sharvari; Heiman, David I.; Henn, Matthew R.; Kodira, Chinnappa D.; León-Narváez, Henry; Longo, Larissa V. G.; Ma, Li-Jun; Malavazi, Iran; Matsuo, Alisson L.; Morais, Flavia V.; Pereira, Maristela; Rodríguez-Brito, Sabrina; Sakthikumar, Sharadha; Salem-Izacc, Silvia M.; Sykes, Sean M.; Teixeira, Marcus Melo; Vallejo, Milene C.; Walter, Maria Emília Machado Telles; Yandava, Chandri; Young, Sarah; Zeng, Qiandong; Zucker, Jeremy; Felipe, Maria Sueli; Goldman, Gustavo H.; Haas, Brian J.; McEwen, Juan G.; Nino-Vega, Gustavo; Puccia, Rosana; San-Blas, Gioconda; Soares, Celia Maria de Almeida; Birren, Bruce W.; Cuomo, Christina A.
Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of
Oliveira, Juliana Velasco de Castro; Dos Santos, Renato Augusto Corrêa; Borges, Thuanny A; Riaño-Pachón, Diego Mauricio; Goldman, Gustavo Henrique
Here, we present the nuclear and mitochondrial genome sequences of Pseudozyma brasiliensis sp. nov. strain GHG001. P. brasiliensis sp. nov. is the closest relative of Pseudozyma vetiver. P. brasiliensis sp. nov. is capable of growing on xylose or xylan as a sole carbon source and has great biotechnological potential.
Oliveira, Juliana Velasco de Castro; dos Santos, Renato Augusto Corrêa; Borges, Thuanny A.
Here, we present the nuclear and mitochondrial genome sequences of Pseudozyma brasiliensis sp. nov. strain GHG001. P. brasiliensis sp. nov. is the closest relative of Pseudozyma vetiver. P. brasiliensis sp. nov. is capable of growing on xylose or xylan as a sole carbon source and has great biotechnological potential. PMID:24356824
Costa, Jane; Bargues, Maria Dolores; Neiva, Vanessa Lima; Lawrence, Gena G; Gumiel, Marcia; Oliveira, Genova; Cabello, Pedro; Lima, Marli Maria; Dotson, Ellen; Provance, David William; Almeida, Carlos Eduardo; Mateo, Lucia; Mas-Coma, Santiago; Dujardin, Jean Pierre
Triatoma brasiliensis macromelasoma occurs in Pernambuco state, Brazil, which is situated between the distribution areas of Triatoma brasiliensis brasiliensis (north) and Triatoma juazeirensis (south). T. b. macromelasoma displays greater variations in its chromatic phenotype than either T. b. brasiliensis or T. juazeirensis, and patterns reminiscent of one or the other. Experimental crosses from each of these members of the T. brasiliensis species complex generated fertile offspring suggesting that viable hybrids could be present in nature, despite their significant genetic distances. Considering the geographical position of occurrence of the T. b. macromelasoma (in Pernambuco) it was proposed to be an area capable of supporting natural hybridization between T. b. brasiliensis and T. juazeirensis. Since phenotypic variability is expected, this study investigated the existence of intermediate chromatic phenotypes for T. b. macromelasoma in various locations in areas between the T. b. brasiliensis and T. juazeirensis occurrences. Thirteen different color patterns were for the first time characterized and nine of those displayed intermediate phenotypes. Molecular analysis performed using ribosomal DNA intergenic region, grouped all within the T. brasiliensis complex. The intermediate chromatic phenotypes, molecular analysis and experimental crosses all support the distinction of a zone of hybridization that gave rise to the T. b. macromelasoma through homoploidal evolution.
Pereira, L L V; Alevi, K C C; Moreira, F F F; Barbosa, J F; Silistino-Souza, E R; Silva Júnior, F C; Souza-Firmino, T S; Banho, C A; Itoyama, M M
Few cytogenetic studies have been undertaken using aquatic heteropterans and the nucleolar behavior of these insects has been described in only four species, Limnogonus aduncus, Brachymetra albinerva, Halobatopsis platensis, and Cylindrostethus palmaris. The nucleolus is a cellular structure related to biosynthetic activity and it exhibits a peculiar behavior in the heteropterans of the Triatominae subfamily; it persists during all stages of meiosis. Thus, this study aims to analyze spermatogenesis in Martarega brasiliensis, with an emphasis on nucleolar behavior. Twenty M. brasiliensis adult males were used and collected from the Municipal reservoir in the city of São José do Rio Preto, São Paulo, Brazil. The species were fixed in methanol:acetic acid (3:1), then dissected, and the testicles were extracted, torn apart, and impregnated with silver ions. During prophase, the nuclei of M. brasiliensis were composed of the nucleolus and nucleolar corpuscles, which varied in number from one to four, emphasizing that this insect has great synthetic activity during meiosis. The analysis of cells in metaphase I showed that M. brasiliensis presents a nucleolar organizing region in at least one autosome. Furthermore, the phenomenon of nucleolar persistence was not observed. All spermatids presented nucleolar markings that varied in number and position according to the stage of elongation. Moreover, it was also possible to highlight the presence of a vesicle in spermatids. Thus, this paper describes the nucleolar behavior of M. brasiliensis and highlights important characteristics during spermatogenesis, thus, increasing the knowledge about the biology of these aquatic heteropterans.
Sanchotene, Karine Ortiz; Madrid, Isabel Martins; Klafke, Gabriel Baracy; Bergamashi, Mariana; Della Terra, Paula Portella; Rodrigues, Anderson Messias; de Camargo, Zoilo Pires; Xavier, Melissa Orzechowski
Sporotrichosis is the main subcutaneous mycosis in Brazil, and is caused by Sporothrix schenckii and allied species. Sporothrix propagules present on soil and plant debris may be traumatically inoculated into the cutaneous/ subcutaneous tissues of the warm-blooded host. An alternative route involves direct animal-animal and animal-human transmissions through deep scratches and bites of diseased cats. Sporotrichosis is much more common than previously appreciated with several cases emerging over the years especially in South and Southeast Brazil. We conducted an epidemiological surveillance in endemic areas of feline sporotrichosis in the southern region of Rio Grande do Sul state, Brazil. Over the last 5-year period the number of feline sporotrichosis in Rio Grande increased from 0.75 new cases per month in 2010 to 3.33 cases per month in 2014. The wide geographic distribution of diagnosed cases highlights the dynamics of Sporothrix transmission across urban areas with high population density. Molecular identification down to species level by PCR-RFLP of cat-transmitted Sporothrix revealed the emergence of the clonal offshoot S. brasiliensis during feline outbreaks; this scenario is similar to the epidemics taking place in the metropolitan areas of Rio de Janeiro and São Paulo. Controlling and preventing sporotrichosis outbreaks are essential steps to managing the disease among humans and animals.
Berthelot, Karine; Peruch, Frédéric; Lecomte, Sophie
Hevein, from Hevea brasiliensis (rubber tree), was identified in 1960. It is the most abundant soluble protein (22%) found in latex. Hevein is formed from a larger protein called prohevein. The 187 amino-acid prohevein is cleaved into two fragments: the N-terminal 43 amino-acid hevein, a lectin bearing a chitin-binding motif with antifungal properties, and a C-terminal domain (C-ter), which possesses amyloid properties. Hevein-like proteins are also widely represented in the plant kingdom and belong to a larger family related to stress and pathogenic responses. During the last 55 years, these proteins have attracted the interest of numerous specialists from the fields of plant physiology, genetics, molecular and structural biology, and physico-chemistry to allergology. This review highlights various aspects of hevein, prohevein, and C-ter from the point of view of these various fields, and examines their potential roles in latex as well as their beneficial and negative biological effects (e.g. wound sealing and resistance to pathogens which is mediated by agglutination, antimicrobial activity, and/or allergenicity). It covers results and observations from 1960 up to the most recent research.
Schmidt, S; Thaller, J
Auditory thresholds improve with increasing signal duration within the maximum integration time of the auditory system, a phenomenon called temporal summation. The temporal summation function is a basic characteristic of particular relevance for bat sonar, as it determines the ability to detect targets with short echolocation calls. Temporal summation was studied in 6 Mexican free-tailed bats (Tadarida brasiliensis) in a forced two-choice behavioural test. Masked auditory thresholds for 40-kHz test tone pulses with durations between 2 ms and 400 ms were determined in broadband noise of two different spectrum levels (-18 dB, +17 dB). At both masker levels, thresholds decreased by considerably more than 10 dB per decade of duration. The time constants of the summation functions, which are a measure of the maximum integration time, shortened significantly with increasing masker level from 62 ms to 14 ms. The steep summation functions are only partly accounted for by spectral splatter. This suggests that the bats are capable of a neural overintegration of sound intensity. Finally, it is shown that such short time constants are typical for echolocating animals, and the implications of the found summation functions for echolocation are considered.
Varga, János; Kocsubé, Sándor; Tóth, Beáta; Frisvad, Jens C; Perrone, Giancarlo; Susca, Antonia; Meijer, Martin; Samson, Robert A
A novel species, Aspergillus brasiliensis sp. nov., is described within Aspergillus section Nigri. This species can be distinguished from other black aspergilli based on intergenic transcribed region, beta-tubulin and calmodulin gene sequences, by amplified fragment length polymorphism analysis and by extrolite profiles. A. brasiliensis isolates produced naphtho-gamma-pyrones, tensidol A and B and pyrophen in common with Aspergillus niger and Aspergillus tubingensis, but also several unique compounds, justifying their treatment as representing a separate species. None of the isolates were found to produce ochratoxin A, kotanins, funalenone or pyranonigrins. The novel species was most closely related to A. niger, and was isolated from soil from Brazil, Australia, USA and The Netherlands, and from grape berries from Portugal. The type strain of Aspergillus brasiliensis sp. nov. is CBS 101740(T) (=IMI 381727(T)=IBT 21946(T)).
Freitas, Dayvison F S; Santos, Suelen S; Almeida-Paes, Rodrigo; de Oliveira, Manoel M E; do Valle, Antonio C F; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely M; Nosanchuk, Joshua D
The metropolitan region of Rio de Janeiro is hyperendemic for cat-associated sporotrichosis. This study aimed to assess the virulence of serial Sporothrix isolates from a 61-year-old male patient with chronic, destructive disseminated sporotrichosis. Five Sporothrix isolates were cultured from skin exudates and bone samples over a 5-year period, and all were molecularly identified as Sporothrix brasiliensis. The final isolate was significantly more virulent in Galleria mellonella larvae compared to earlier isolates. We conclude that S. brasiliensis has the capacity to increase in virulence in vivo. This finding is significant to clinicians caring for individuals with S. brasiliensis disease and it suggests that further studies are needed to identify the mechanisms underlying pathogenicity enhancement during chronic disease.
de Oliveira, Andrea L; Eler, G Jacklin; Bracht, Adelar; Peralta, Rosane M
The effects of a hydroalcoholic extract of Agaricus brasiliensis (A. blazei) on functional parameters in the perfused rat liver were examined with emphasis on its content of nucleotides and nucleosides. Several nucleosides and nucleotides were identified in the A. brasiliensis extract, which was active on several liver functions. A significant part of the effects is the result of the purinergic action of nucleosides and nucleotides: pressure increment, glycogenolysis stimulation, transient inhibition of oxygen consumption, and redox state changes. Other phenomena such as the stimulation of gluconeogenesis, ureogenesis, and oxygen consumption are more likely consequences of the metabolic transformation of substrates contained within the extract, especially amino acids. It seems apparent that consumption of A. brasiliensis represents not only the ingestion of metabolic precursors but also the ingestion of substances that, even at low concentrations, can exert important signaling functions in the liver as well as in the organism as a whole.
Rua, Cintia P J; Trindade-Silva, Amaro E; Appolinario, Luciana R; Venas, Tainá M; Garcia, Gizele D; Carvalho, Lucas S; Lima, Alinne; Kruger, Ricardo; Pereira, Renato C; Berlinck, Roberto G S; Valle, Rogério A B; Thompson, Cristiane C; Thompson, Fabiano
Marine sponges are the oldest Metazoa, very often presenting a complex microbial consortium. Such is the case of the marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro State, Brazil. In this investigation we characterized the diversity of some of the culturable heterotrophic bacteria living in association with A. brasiliensis and determined their antimicrobial activity. The genera Endozoicomonas (N = 32), Bacillus (N = 26), Shewanella (N = 17), Pseudovibrio (N = 12), and Ruegeria (N = 8) were dominant among the recovered isolates, corresponding to 97% of all isolates. Approximately one third of the isolates living in association with A. brasiliensis produced antibiotics that inhibited the growth of Bacillus subtilis, suggesting that bacteria associated with this sponge play a role in its health.
Rua, Cintia P.J.; Trindade-Silva, Amaro E.; Appolinario, Luciana R.; Venas, Tainá M.; Garcia, Gizele D.; Carvalho, Lucas S.; Lima, Alinne; Kruger, Ricardo; Pereira, Renato C.; Berlinck, Roberto G.S.; Valle, Rogério A.B.; Thompson, Cristiane C.
Marine sponges are the oldest Metazoa, very often presenting a complex microbial consortium. Such is the case of the marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro State, Brazil. In this investigation we characterized the diversity of some of the culturable heterotrophic bacteria living in association with A. brasiliensis and determined their antimicrobial activity. The genera Endozoicomonas (N = 32), Bacillus (N = 26), Shewanella (N = 17), Pseudovibrio (N = 12), and Ruegeria (N = 8) were dominant among the recovered isolates, corresponding to 97% of all isolates. Approximately one third of the isolates living in association with A. brasiliensis produced antibiotics that inhibited the growth of Bacillus subtilis, suggesting that bacteria associated with this sponge play a role in its health. PMID:25024903
Garcia, D M; Marmontel, M; Rosas, F W; Santos, F R
The giant otter (Pteronura brasiliensis) is an aquatic mammal of the Mustelidae family, endemic to South America. Its original distribution corresponds to the region from the Guyanas to Central-North Argentina, but it is extinct or on the verge of extinction in most of its historical range. Currently, the species is considered endangered by the World Conservation Union (IUCN). Based on its geographic distribution in the South American continent and on some morphological characters, two subspecies were suggested: P. brasiliensis brasiliensis, occurring in the Amazon and Orinoco River Basins, and P. brasiliensis paranensis, in the Paraná and Paraguai River Basins. However, there is no consensus on assuming this subspecies division and no detailed studies have been carried out to elucidate this question. This study aims to evaluate the genetic diversity and population structure of Pteronura brasiliensis along its range in Brazil to check the possibility of the existence of two distinct subspecies using also a reciprocal monophyly criterion. We analyzed the control region, and the Cytochrome b and Cytochrome c Oxidase subunit I genes of the mitochondrial DNA in several giant otter populations from the Amazon and Paraguai River Basins. Analyses have indicated some degree of geographic correlation and a high level of inter-population divergence, although the subspecies division is not highly supported. As we observed strong population structure, we cannot rule out the existence of further divisions shaping the species distribution. The results suggest that a more complex population structure occurs in P. brasiliensis, and the conservation practice should concentrate on preserving all remaining local populations.
Zhang, Lan; Yuan, Bo; Wang, HuiPing; Gao, Ya
The present study was designed to investigate the effect of Agaricus brasiliensis extract (ABE) on phenylhydrazine-induced neonatal jaundice in rats. Administration of ABE dose-dependently reduced the elevated bilirubin level induced by phenylhydrazine. It can be somewhat supported from the results of in vitro bilirubin degradation experiment. ABE treatment also reduced the total antioxidant status (TAOS), cascade O2−/SOD, level of NF-κB protein, and adrenomedullin (AM). Overall, the results of this study demonstrated that Agaricus brasiliensis extract may be beneficial to reducing bilirubin level without causing hepatotoxicity in neonatal jaundice. PMID:25883968
Fukuwatari, Yasushi; Okumura, Ko; Takeda, Kazuyoshi; Ishibashi, Ken-ichi; Furukawa, Mai; Ohno, Naohito; Mori, Kazu; Gao, Ming; Motoi, Masuro
We performed studies on murine models and human volunteers to examine the immunoenhancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazei). Antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit body is useful as a health-promoting food. PMID:18604247
Singh, Adya P; Wi, Seung Gon; Chung, Gap Chae; Kim, Yoon Soo; Kang, Hunseung
Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.
Orofino-Costa, Rosane; Unterstell, Natasha; Carlos Gripp, Alexandre; de Macedo, Priscila Marques; Brota, Arles; Dias, Emylli; de Melo Teixeira, Marcus; Felipe, Maria Sueli; Bernardes-Engemann, Andréa R; Lopes-Bezerra, Leila Maria
A 32-year-old HIV negative male presented with multiple pulmonary cavitation and skin abscesses up to 15 cm in diameter mimicking tuberculosis. Sporothrix brasiliensis was isolated and patient responded well to amphotericin B followed by itraconazole, except the skin lesions that had to be surgical drained to obtain cure. PMID:24432220
Training is a critical part of aphid (Hemiptera: Aphididae) identification. This video provides provides training to identify the palm aphid, Cerataphis brasiliensis, using a compound microscope and an electronic identification key called “LUCID.” The video demonstrates key morphological structures...
Borba-Santos, Luana Pereira; Rodrigues, Anderson Messias; Gagini, Thalita Braga; Fernandes, Geisa Ferreira; Castro, Rafaela; de Camargo, Zoilo Pires; Nucci, Marcio; Lopes-Bezerra, Leila Maria; Ishida, Kelly; Rozental, Sonia
The in vitro activity of the antifungal agents amphotericin B (AMB), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), and terbinafine (TRB) against 32 Brazilian isolates of Sporothrix brasiliensis, including 16 isolates from a recent (2011-2012) epidemic in Rio de Janeiro state, was examined. We describe and genotype new isolates and clustered them with 16 older (from 2004 or earlier) S. brasiliensis isolates by phylogenetic analysis. We tested both the yeast and the mycelium form of all isolates using broth microdilution methods based on the reference protocols M38-A2 and M27-A3 (recommended by the Clinical and Laboratory Standards Institute). Considering minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), TRB was found to be the most active drug in vitro for both fungal forms, followed by PSC. Several isolates showed high MICs for AMB and/or ITC, which are currently used as first-line therapy for sporotrichosis. VRC displayed very low activity against S. brasiliensis isolates. The primary morphological modification observed on treated yeasts by transmission electron microscopy analysis was changes in cell wall. Our results indicate that TRB is the antifungal with the best in vitro activity against S. brasiliensis and support the use of TRB as a promising option for the treatment of cutaneous and/or lymphocutaneous sporotrichosis.
Cibulski, Samuel Paulo; Teixeira, Thais Fumaco; de Sales Lima, Francisco Esmaile; do Santos, Helton Fernandes; Franco, Ana Claudia
Using metagenomic approaches, we identified a novel Torque teno virus from Brazilian free-tailed bats (Tadarida brasiliensis) (TT-TbV). The TT-TbV genome and deduced protein sequences share extremely low identity with known anelloviruses. Due to a high degree of phylogenetic divergence, such putative virus could not be allocated into any Anelloviridae genera. PMID:25359906
Bartsch, E; Schmidt, S
Echolocating bats hunting flying insects discriminate complex temporal patterns of acoustic stimuli. For bats using frequency modulated sonar calls (FM bats), there are no behavioral data on the perception of sinusoidally frequency modulated (SFM) stimuli. Discrimination performance for SFM stimuli of varying modulation depth was measured in 4 Tadarida brasiliensis in a two-alternative, forced choice procedure. A center frequency of 40 kHz was modulated with rates between 10 and 2000 Hz. It was found that discrimination performance improved from a mean threshold modulation depth of 3.05 kHz at a modulation rate of 2000 Hz to 1.58 kHz at a modulation rate of 10 Hz. Psychoacoustical modulation depth thresholds of T. brasiliensis are thus distinctly larger than those observed in bat species emitting constant frequency (CF) components followed by an FM-sweep, in active echolocation experiments. The modulation thresholds of T. brasiliensis are discussed in connection with the ability of bats to discriminate insect wingbeats. A comparison between non-echolocating mammals and the FM bat T. brasiliensis shows that the ability to echolocate is not reflected in the modulation thresholds.
Cibulski, Samuel Paulo; Teixeira, Thais Fumaco; de Sales Lima, Francisco Esmaile; do Santos, Helton Fernandes; Franco, Ana Claudia; Roehe, Paulo Michel
Using metagenomic approaches, we identified a novel Torque teno virus from Brazilian free-tailed bats (Tadarida brasiliensis) (TT-TbV). The TT-TbV genome and deduced protein sequences share extremely low identity with known anelloviruses. Due to a high degree of phylogenetic divergence, such putative virus could not be allocated into any Anelloviridae genera.
Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.
Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla Dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida; Pereira, Maristela
Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.
Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida
Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. PMID:26124176
Lemes, Paola C R; Loeb, Marina V; Santificetur, César; Melo, Marcelo R S
Urophycis Gill 1864 is a genus of demersal fish composed by eight valid species with anti-tropical distributions in the western Atlantic. Only two species occur in the South Atlantic: U. brasiliensis (Kaup 1858) and U. mystacea Miranda Ribeiro 1903. These species have similar ranges from southeastern Brazil to northern Argentina, but U. brasiliensis occurs in more shallow waters than U. mystacea. Both species are important fishery resources in southern Brazil and Uruguay. Herein, we redescribe U. brasiliensis based on comparison of types and additional specimens, including the description of the Sagitta otolith, formerly place U. latus Miranda Ribeiro 1903 as a junior synonym of U. brasiliensis, and provide an updated map of distribution.
Hubel, Tatjana Y; Hristov, Nickolay I; Swartz, Sharon M; Breuer, Kenneth S
To date, wake measurements using particle image velocimetry (PIV) of bats in flight have studied only three bat species, all fruit and nectar feeders. In this study, we present the first wake structure analysis for an insectivorous bat. Tadarida brasiliensis, the Brazilian free-tailed bat, is an aerial hunter that annually migrates long distances and also differs strikingly from the previously investigated species morphologically. We compare the aerodynamics of T. brasiliensis with those of other, frugivorous bats and with common swifts, Apus apus, a bird with wing morphology, kinematics and flight ecology similar to that of these bats. The comparison reveals that, for the range of speeds evaluated, the cyclical pattern of aerodynamic forces associated with a wingbeat shows more similarities between T. brasiliensis and A. apus than between T. brasiliensis and other frugivorous bats.
Canals, Mauricio; Atala, Cristian; Olivares, Ricardo; Guajardo, Francisco; Figueroa, Daniela P; Sabat, Pablo; Rosenmann, Mario
We studied structure and function of the respiratory system in the bat Tadarida brasiliensis and compared it with those of two species of rodents, Abrothrix andinus and A. olivaceus. Tadarida brasiliensis had lower resting oxygen consumption, but higher maximum oxygen consumption and aerobic scope, than the rodents. The blood-gas barrier of the bat was thinner and its relative lung size was larger; however, alveolar surface density was similar among the three species. In consequence, T. brasiliensis has an oxygen diffusion capacity two or three times higher than that of the rodents. In Tadarida brasiliensis the characteristics of the lung were accompanied by geometrical changes in the proximal airway, such as high physical optimization as a consequence of small variations in the symmetry and the scaling ratio of the bronchial diameters. These may constitute an efficient way to save energy in respiratory mechanics and are the first report of airway adjustments to decrease entropy generation in bats.
Martins, Aline R.; Abreu, Aluana G.; Bajay, Miklos M.; Villela, Priscilla M. S.; Batista, Carlos E. A.; Monteiro, Mariza; Alves-Pereira, Alessandro; Figueira, Glyn M.; Pinheiro, José B.; Appezzato-da-Glória, Beatriz; Zucchi, Maria I.
• Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers were developed for Smilax brasiliensis, which is popularly known as sarsaparilla and used in folk medicine as a tonic, antirheumatic, and antisyphilitic. Smilax brasiliensis is sold in Brazilian pharmacies, and its origin and effectiveness are not subject to quality control. • Methods and Results: Using a protocol for genomic library enrichment, primer pairs were developed for 26 microsatellite loci and validated in 17 accessions of S. brasiliensis. Thirteen loci were polymorphic and four were monomorphic. The primers successfully amplified alleles in the congeners S. campestris, S. cissoides, S. fluminensis, S. goyazana, S. polyantha, S. quinquenervia, S. rufescens, S. subsessiliflora, and S. syphilitica. • Conclusions: The new SSR markers described herein are informative tools for genetic diversity and gene flow studies in S. brasiliensis and several congeners. PMID:25202555
Steece, R; Altenbach, J S
Adult female and juvenile Mexican free-tailed bats (Tadarida brasiliensis mexicana) were collected bimonthly at Lava Cave, New Mexico from May through September. The purpose of this study was to examine the prevalence of active rabies infection as well as to determine individual immune status in these hosts. All bats were bled and examined for rabies antibody (total antibody versus IgM) utilizing a modified serum neutralization test. The brains were removed and examined by the fluorescent rabies antibody (FRA) test. No significant difference was observed in the number of adults with rabies neutralizing antibody (total) over the study period. Significant differences in rabies neutralizing antibody (total) were observed among the juveniles sampled during July and August. The number of adults with IgM specific antibody was low (15 of 750, 2%) and did not fluctuate significantly. However, the number of juveniles with IgM antibody did show increased levels in August and September. The number of adults positive by the FRA was low (4 of 750, less than 1%) and did not appear to fluctuate significantly over the study period. The number of juveniles positive by the FRA was three and one-half times higher than observed for the adults (14 of 600, 2%). These results indicate that the Mexican free-tailed bat appears to be exposed to rabies virus shortly after birth as evident by its immune status. The low prevalence (4 of 750, less than 1%) of active infection as determined by the FRA and mouse inoculation and the high prevalence (514 of 750, 69%) of IgG antibody in the adult females indicate that the Mexican free-tailed bat recovers from rabies virus infection.
Bunyatang, Orawan; Chirapongsatonkul, Nion; Bangrak, Phuwadol; Henry, Robert; Churngchow, Nunta
A novel cDNA encoding a bi-functional α-amylase/subtilisin inhibitor (HbASI) was isolated from rubber (Hevea brasiliensis) leaves cultivar RRIM600. The HbASI had strong homology with the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Its putative amino acid sequence was similar to that of the α-amylase/subtilisin inhibitor from Ricinus communis (72% identity). Genomic sequencing indicated that the HbASI gene contained no introns. The messenger RNA of HbASI was detected in leaf, hypocotyl and root. The recombinant HbASI expressed extracellularly in Pichia pastoris exhibited inhibitory activity against α-amylase from Aspergillus oryzae, trypsin and subtilisin A. The HbASI gene was induced in the rubber leaves infected with a rubber tree pathogen, Phytophthora palmivora. It was also enhanced by salicylic acid (SA) treatment and mechanical wounding. In addition, the biological activity of the HbASI protein involving in the plant defence responses was also investigated. The HbASI at a concentration of 0.16 mg mL(-1) could inhibit the mycelium growth of P. palmivora. These data suggested that the HbASI protein might play a crucial role in defence against pathogen of rubber trees.
Peng, Shi-Qing; Wu, Kun-Xin; Huang, Gui-Xiu; Chen, Shou-Cai
Tapping panel dryness (TPD) is a complex physiological syndrome found widely in rubber tree (Hevea brasiliensis) plantations that causes severe yield loss in natural rubber-producing countries. In an earlier study, we confirmed that there is a negative correlation between HbMyb1 expression and TPD severity. To further investigate the function of HbMyb1 in TPD, HbMyb1 was over-expressed in tobacco controlled by a CaMV 35S promoter. In transgenic plants expressing HbMyb1, cell death induced by UV-B irradiation, paraquat and the hypersensitive reaction to necrotrophic fungal infection (Botrytis cinerea) was suppressed with a close correlation between HbMyb1 protein levels and the extent of suppression. In addition the nuclear condensation and degradation were observed in laticifer cells of TPD trees, while the nucleus of laticifer cells of healthy trees was morphologically normal. On the basis of the results described above, we propose that HbMyb1 maybe suppress stress induced cell death in rubber trees.
da Silva, Marcos Zatti; Sato, Mário Eidi; de Oliveira, Carlos Amadeu Leite; Nicastro, Roberto Lomba
Brevipalpus phoenicis (Geijskes) is associated with the transmission of Citrus leprosis which is considered the main viral disease for the Brazilian citrus production. Mites of the families Stigmaeidae and Phytoseiidae coexist in various agricultural crops, often promoting the biological control of pest mites. The aim of this work was to study the interactions of Neoseiulus californicus (McGregor) (Phytoseiidae) and Agistemus brasiliensis Matioli, Ueckermann & Oliveira (Stigmaeidae), in the presence or absence of B. phoenicis. Two experiments were carried out. In the first, a N. californicus female was placed in each leaf disc arena, with eggs of B. phoenicis and A. brasiliensis as food sources. In the second, an A. brasiliensis female was placed in each arena, with eggs of B. phoenicis and N. californicus as food sources. Adults of both predators were able to consume both types of eggs available as food sources, but they fed on considerably higher proportions of B. phoenicis than on eggs of the predator. Eggs of A. brasiliensis were not a suitable food source for N. californicus, which produced only 0.1 egg per female per day when only eggs of that species were present in the experimental unit. The results suggest that eggs of N. californicus were a suitable food source for A. brasiliensis, which oviposited 1.12 eggs per day, when only eggs of N. californicus were provided to the stigmaeid mite. The possible interactions among N. californicus, A. brasiliensis and B. phoenicis in citrus orchards are discussed.
Castro, Rafaela A; Kubitschek-Barreira, Paula H; Teixeira, Pedro A C; Sanches, Glenda F; Teixeira, Marcus M; Quintella, Leonardo P; Almeida, Sandro R; Costa, Rosane O; Camargo, Zoilo P; Felipe, Maria S S; de Souza, Wanderley; Lopes-Bezerra, Leila M
Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.
Valença-Barbosa, Carolina; Lima, Marli M; Sarquis, Otília; Bezerra, Claudia M; Abad-Franch, Fernando
Triatoma brasiliensis is the most important vector of Chagas disease in the Caatinga eco-region of northeastern Brazil. Wild T. brasiliensis populations have been reported only from rocky outcrops. However, this species frequently infests/re-infests houses in rock-free sedimentary lowlands. We therefore hypothesized that it should also occupy other natural ecotopes. We show that a common Caatinga cactus, Pilosocereus gounellei, locally known as xiquexique, often harbors T. brasiliensis breeding colonies apparently associated with rodents (n = 44 cacti, infestation rate = 47.7%, 157 bugs captured). Our findings suggest that infested cacti might be involved in house re-infestation by T. brasiliensis in the Caatinga region.
Shanmugaprakash, M; Kirthika, J; Ragupathy, J; Nilanee, K; Manickam, A
Statistics based optimization, Plackett-Burman design (PBD) and response surface methodology (RSM) were employed to screen and optimize the media components for the production of naringinase from Aspergillus brasiliensis MTCC 1344, using solid state fermentation. Cassava waste (CW) was used as both the solid support and carbon source for the growth of A. brasiliensis. Based on the positive influence of the Pareto chart obtained from PBD on naringinase activity, three media components--maltose, peptone and calcium chloride were screened. Box-Behnken design (BBD) was employed using these three factors at three levels, for further optimization, and the second order polynomial equation was derived, based on the experimental data. Derringer's desired function methodology showed that the concentrations of maltose (7.74 g/L), peptone (4.19 g/L) and calcium chloride (7.63 mM) were the optimal levels for maximal naringinase activity (889.91 U/mg) which were validated through experiments.
Bittencourt, Flora; Alves, Jackeline S.; Gaiotto, Fernanda A.
Premise of the study: We developed microsatellite markers for Carpotroche brasiliensis (Flacourtiaceae), a dioecious tree that is used as a food resource by midsize animals of the Brazilian fauna. Methods and Results: We designed 30 primer pairs using next-generation sequencing and classified 25 pairs as polymorphic. Observed heterozygosity ranged from 0.5 to 1.0, and expected heterozygosity ranged from 0.418 to 0.907. The combined probability of exclusion was greater than 0.999 and the combined probability of identity was less than 0.001, indicating that these microsatellites are appropriate for investigations of genetic structure, individual identification, and paternity testing. Conclusions: The developed molecular tools may contribute to future studies of population genetics, answering ecological and evolutionary questions regarding efficient conservation strategies for C. brasiliensis. PMID:26697275
Tanaka, Y; Komaki, H; Yazawa, K; Mikami, Y; Nemoto, A; Tojyo, T; Kadowaki, K; Shigemori, H; Kobayashi, J
A new 32-membered macrolide antibiotic, brasilinolide A was isolated from the fermentation broth of Nocardia sp. IFM 0406. The producer was identified as Nocardia brasiliensis. The antibiotic was only active against Aspergillus niger, but not active against other fungi including yeasts as well as other filamentous like fungi and bacteria. Brasilinolide A exerted an immunosuppressive activity in the assay system of a mixed lymphocyte reaction (MLR).
Batista, V S P; Fernandes, F A; Cordeiro-Estrela, P; Sarquis, O; Lima, M M
Triatoma brasiliensis (Hemiptera: Reduviidae) is an important vector of Chagas' disease in both sylvatic and peridomestic ecotopes. Discriminating between these populations of Triatominae has been proposed as a means of investigating re-infestation rates of human dwellings. Geometric morphometrics have been widely applied in the study of Triatominae polymorphisms at species and population levels. This study characterizes morphometric differences between sylvatic and peridomestic populations, as well as between sexes in T. brasiliensis specimens from Jaguaruana, Ceará, in northeastern Brazil. No differences in either the shape or size of the cephalic capsule were apparent between sexes or ecotopes. However, the wings showed differentiation in shape and size. Sexual dimorphism was detected, with females presenting significantly higher values and conformations. Size differentiation was also evident, with sylvatic specimens being generally larger than peridomestic examples. These results indicate that differences in the wings of T. brasiliensis may be related to the existence of phenotypic plasticity, and variations in size and shape may be associated with different ecotopes, possibly as a result of conditions in each micro-habitat, such as temperature, relative humidity, food supply and density.
Waller, Stefanie Bressan; Madrid, Isabel Martins; Silva, Anna Luiza; Dias de Castro, Luciana Laitano; Cleff, Marlete Brum; Ferraz, Vanny; Meireles, Mário Carlos Araújo; Zanette, Régis; de Mello, João Roberto Braga
This study evaluated the chemical, cytotoxic and anti-Sporothrix brasiliensis properties of commercial essential oils of rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.) and marjoram (Origanum majorana L.). Chemical composition of the oils was identified through gas chromatography with flame ionization detector, and cytotoxicity was performed through MTT assay in VERO cell line. Anti-S. brasiliensis activity was performed according to the CLSI M38-A2 guidelines using isolates obtained from cats and dogs. The major compounds found were carvacrol in the oregano oil (73.9 %) and 1,8-cineole in rosemary and marjoram oils (49.4 and 20.9 %, respectively). All S. brasiliensis isolates were susceptible to the plant oils, including itraconazole-resistant ones. Marjoram and rosemary oils showed MIC90 of 0.56 and 1.12 mg ml(-1), and MFC90 of 4.5 and 9 mg ml(-1), respectively. For oregano oil, a strong antifungal activity was observed with MIC90 and MFC90 values ≤0.07 mg ml(-1). The weakest cytotoxicity was observed for rosemary oil. Further studies should be undertaken to evaluate the safety and efficacy of these essential oils in sporotrichosis.
Yurkiv, Borys; Wasser, Solomon P; Nevo, Eviatar; Sybirna, Nataliya O
With diabetes mellitus and increased glucose concentrations, the mitochondria electron transport chain is disrupted, superoxide anions are overproduced, and oxidative stress develops in cells. Thus, preventing oxidative stress can produce a decrease in the antioxidant system activity and an increase in apoptosis in immune cells. The application of medicinal mushrooms is a new possible approach to diabetes mellitus treatment. Therefore, the aim of this work was to investigate the influence of administration of the medicinal mushrooms Agaricus brasiliensis and Ganoderma lucidum on antioxidant enzyme activity in rat leukocytes. Wistar outbred white rats were used in the study. Streptozotocin was intraperitoneally injected once at a dose of 50 mg/kg body weight. Mushroom preparations were orally administered at a dose of 1 g/kg/day for 2 weeks. This revealed that in diabetes mellitus, the level of antioxidant enzyme activity is significantly decreased compared with control values, whereas the levels of lipid peroxidation is increased; this manifested in an increase in the amount of thiobarbituric acid reactive substances (TBARS). The medicinal mushrooms' administration is accompanied by an increase in antioxidant enzyme activity to control values and is even higher in the case of A. brasiliensis administration when compared with the diabetic group. As for the indicators of lipid peroxidation under mushroom administration of A. brasiliensis and G. lucidum, we observed a significant decrease of TBARS levels compared with the diabetic group. Increased activity of antioxidant enzymes and reduction of TBARS level indicate pronounced antioxidant properties of studied mushrooms.
Aureliano, Tito; Ghilardi, Aline M.; Guilherme, Edson; Souza-Filho, Jonas P.; Cavalcanti, Mauro; Riff, Douglas
Purussaurus brasiliensis thrived in the northwestern portion of South America during the Late Miocene. Although substantial material has been recovered since its early discovery, this fossil crocodilian can still be considered as very poorly understood. In the present work, we used regression equations based on modern crocodilians to present novel details about the morphometry, bite-force and paleobiology of this species. According to our results, an adult Purussaurus brasiliensis was estimated to reach around 12.5 m in length, weighing around 8.4 metric tons, with a mean daily food intake of 40.6 kg. It was capable of generating sustained bite forces of 69,000 N (around 7 metric tons-force). The extreme size and strength reached by this animal seems to have allowed it to include a wide range of prey in its diet, making it a top predator in its ecosystem. As an adult, it would have preyed upon large to very large vertebrates, and, being unmatched by any other carnivore, it avoided competition. The evolution of a large body size granted P. brasiliensis many advantages, but it may also have led to its vulnerability. The constantly changing environment on a large geological scale may have reduced its long-term survival, favoring smaller species more resilient to ecological shifts. PMID:25689140
Tong, Zheng; Sun, Yong; Wang, Dan; Wang, Limin; Li, Ling; Meng, Xueru; Feng, Weiqiang; Wurtele, Eve Syrkin; Wang, Xuchu
Latex in the laticiferous cell network of Hevea brasiliensis tree is composed of cytoplasm that synthesizes natural rubber. Ethylene stimulation of the tree bark enhances latex production partly by prolonging the duration of latex flow during the tapping process. Here, we identified an osmotin-like cDNA sequence (HbOsmotin) from H. brasiliensis that belongs to the pathogenesis-related 5 (PR-5) gene family. The HbOsmotin protein is present in the lutoids of latex in H. brasiliensis, whereas in onion epidermal cells, this protein is predominantly distributed around the cell wall, suggesting that it may be secreted from the cytoplasm. We investigated the effects of exogenous ethylene on HbOsmotin transcription and protein accumulation in rubber latex, and further determined the protein function after osmotic stress in Arabidopsis. In regularly tapped trees, HbOsmotin expression was drastically inhibited in rubber latex after tapping, although the expression was subsequently recovered by ethylene stimulation. However, in virgin plants that had never been tapped, exogenous ethylene application slightly decreased HbOsmotin expression. HbOsmotin overexpression in Arabidopsis showed that HbOsmotin reduced the osmotic stress tolerance of the plant, which likely occurred by raising the water potential. These data indicated that HbOsmotin may contribute to osmotic regulation in laticiferous cells.
Kaupert Neto, Antonio Adalberto; Borin, Gustavo Pagotto; Goldman, Gustavo Henrique; Damásio, André Ricardo de Lima; Oliveira, Juliana Velasco de Castro
In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of biofuel production. A novel basidiomycete yeast species, Pseudozyma brasiliensis, was recently discovered. It produces an endo-β-1,4-xylanase with a higher specific activity than other xylanases. This enzyme is essential for the hydrolysis of biomass-derived xylan and has an important role in 2G bioethanol production. In spite of the P. brasiliensis biotechnological potential, there is no information about how it breaks down polysaccharides. For the first time, we characterized the secretome of P. brasiliensis grown on different carbon sources (xylose, xylan, cellobiose and glucose) and also under starvation conditions. The growth and consumption of each carbohydrate and the activity of the CAZymes of culture supernatants were analyzed. The CAZymes found in its secretomes, validated by enzymatic assays, have the potential to hydrolyze xylan, mannan, cellobiose and other polysaccharides. The data show that this yeast is a potential source of hydrolases, which can be used for biomass saccharification.
Santos, Adriana; Ribeiro, José Marcos C.; Lehane, Michael J.; Gontijo, Nelder Figueiredo; Veloso, Artur Botelho; Sant'Anna, Mauricio R.V.; Araujo, Ricardo Nascimento; Grisard, Edmundo C.; Pereira, Marcos Horácio
Triatoma brasiliensis is the most important autochthon vector of Trypanosoma cruzi in Brazil, where it is widely distributed in the semiarid areas of the Northeast. In order to advance the knowledge of the salivary biomolecules of Triatominae, a salivary gland cDNA library of T. brasiliensis was mass sequenced and analyzed. Polypeptides were sequenced by HPLC/Edman degradation experiments. 1,712 cDNA sequences were obtained and grouped in 786 clusters. The housekeeping category had 24.4% and 17.8% of the clusters and sequences, respectively. The putatively secreted category contained 47.1% of the clusters and 68.2% of the sequences. Finally, 28.5% of the clusters, containing 14% of all sequences, were classified as unknown. The sialoma of T. brasiliensis showed a high amount and great variety of different lipocalins (93.8% of secreted proteins). Remarkably, a great number of serine proteases that were not observed in previous blood-sucking sialotranscriptomes were found. Nine Kazal peptides were identified, among them one with high homology to the tabanid vasodilator vasotab, suggesting that the Triatoma vasodilator could be a Kazal protein. PMID:17550826
Eller, Cleiton B; Burgess, Stephen S O; Oliveira, Rafael S
Trees from tropical montane cloud forest (TMCF) display very dynamic patterns of water use. They are capable of downwards water transport towards the soil during leaf-wetting events, likely a consequence of foliar water uptake (FWU), as well as high rates of night-time transpiration (Enight) during drier nights. These two processes might represent important sources of water losses and gains to the plant, but little is known about the environmental factors controlling these water fluxes. We evaluated how contrasting atmospheric and soil water conditions control diurnal, nocturnal and seasonal dynamics of sap flow in Drimys brasiliensis (Miers), a common Neotropical cloud forest species. We monitored the seasonal variation of soil water content, micrometeorological conditions and sap flow of D. brasiliensis trees in the field during wet and dry seasons. We also conducted a greenhouse experiment exposing D. brasiliensis saplings under contrasting soil water conditions to deuterium-labelled fog water. We found that during the night D. brasiliensis possesses heightened stomatal sensitivity to soil drought and vapour pressure deficit, which reduces night-time water loss. Leaf-wetting events had a strong suppressive effect on tree transpiration (E). Foliar water uptake increased in magnitude with drier soil and during longer leaf-wetting events. The difference between diurnal and nocturnal stomatal behaviour in D. brasiliensis could be attributed to an optimization of carbon gain when leaves are dry, as well as minimization of nocturnal water loss. The leaf-wetting events on the other hand seem important to D. brasiliensis water balance, especially during soil droughts, both by suppressing tree transpiration (E) and as a small additional water supply through FWU. Our results suggest that decreases in leaf-wetting events in TMCF might increase D. brasiliensis water loss and decrease its water gains, which could compromise its ecophysiological performance and survival
The soft tick Ornithodoros brasiliensis (Acari: Argasidae) is present in farms along the highlands of Rio Grande do Sul state in southern Brazil. Reports of human parasitism by O. brasiliensis drew the attention of local health authorities. A preliminary epidemiological survey was conducted to ident...
Panti-May, J A; Hernández-Betancourt, S F; Rodríguez-Vivas, R I; Robles, M R
The aim of the present study was to calculate the prevalence and intensity of intestinal helminths in the house mouse (Mus musculus) and the black rat (Rattus rattus) trapped in rural households of Yucatan, Mexico. Sampling was conducted during the rainy season from October to December 2011 and the dry season from January to March 2012. A total of 154 M. musculus and 46 R. rattus were examined, with 84.2% of M. musculus being infected with helminths compared with a significantly lower prevalence of 52.2% in R. rattus (P< 0.01). Adult M. musculus were more likely to be infected with helminths (89%) than subadults (63%) (P< 0.01). Four helminth species were identified: Taenia taeniaeformis larvae, Nippostrongylus brasiliensis, Syphacia muris and Trichuris muris. Nippostrongylus brasiliensis was present more frequently in M. musculus than in R. rattus (P< 0.01) and in adult mice compared to subadults (P< 0.01). Trichuris muris was present only in adult mice. This is the first report of N. brasiliensis, S. muris and T. muris in Yucatan, Mexico, as well as the first to report the presence of N. brasiliensis in M. musculus from Mexico. The helminth fauna of commensal rodents present in households appears to constitute a low potential health risk to local inhabitants; however, it would be advisable to conduct further studies to better understand the public health risk posed by these rodent intestinal helminths.
Uyanoglu, Mustafa; Canbek, Mediha; van Griensven, Leo J L D; Yamac, Mustafa; Senturk, Hakan; Kartkaya, Kazım; Oglakcı, Aysegul; Turgak, Ozge; Kanbak, Gungor
In the present study, the curative effects of crude polysaccharides (PSs) from mushrooms on the symptoms of alcoholic liver injury were investigated. PSs from Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus fruiting bodies were administered by gavage at levels of 100 mg per kg body weight per day for 7 d after the onset of the disease. The caspase-3 activity, mitochondrial membrane potential, mitochondrial outer membrane integrity of the liver tissues of sacrificed rats, and the serum alanine aminotransferase (ALT) levels were determined. In addition, light and transmission electron microscope (TEM) studies were performed for histopathological and cytological evaluations on liver sections. PSs from A. brasiliensis decreased ALT level and mitochondrial membrane potential and increased the outer membrane integrity; microscopic examinations also revealed normal hepatocytes and tissue. On the basis of our data, it can be argued that crude PSs from Agaricus brasiliensis have therapeutic potential for alcoholic liver injury.
Castro, Rafaela A.; Kubitschek-Barreira, Paula H.; Teixeira, Pedro A. C.; Sanches, Glenda F.; Teixeira, Marcus M.; Quintella, Leonardo P.; Almeida, Sandro R.; Costa, Rosane O.; Camargo, Zoilo P.; Felipe, Maria S. S.; de Souza, Wanderley; Lopes-Bezerra, Leila M.
Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes. PMID:24116065
Daflon-Teixeira, Natália Faria; Carvalho-Costa, Filipe Aníbal; Chiang, Ralem Gary; Lima, Marli Maria
The influence of blood meal and mating on Triatoma brasiliensis (Neiva) female fecundity, fertility, life-span and the preoviposition period were investigated under laboratory conditions. Nourishment increased fecundity, fertility and adult lifespan, whereas mating increased fecundity, fertility and decreased the preoviposition period. Females also required more than one mating to reach their full reproductive potential. Results indicate that both nourishment and mating are important in T. brasiliensis proliferation. Such information will help towards developing effective control strategies of this vector of Chagas disease.
Dorigo, T A; Maia-Carneiro, T; Almeida-Gomes, M; Siqueira, C C; Vrcibradic, D; Van Sluys, M; Rocha, C F D
Our study aimed to add information about the diet and endoparasites of Enyalius brasiliensis from an Atlantic Rainforest remnant in the state of Rio de Janeiro, southeastern Brazil. Regarding diet, E. brasiliensis consumed arthropods, with caterpillars and beetles being the most important preys. Regarding helminth parasites, overall prevalence was low (9.5%), with 238 nematodes of the genus Physaloptera found in the stomach of one specimen and one nematode of the genus Rhabdias inside the lung of another. Our results corroborate the observations of previous studies that indicate that lizards of the genus Enyalius tend to feed mainly on relatively large-bodied arthropods and to harbour depauperate endoparasite fauna.
Waller, Stefanie Bressan; Madrid, Isabel Martins; Ferraz, Vanny; Picoli, Tony; Cleff, Marlete Brum; de Faria, Renata Osório; Meireles, Mário Carlos Araújo; de Mello, João Roberto Braga
The study aimed to evaluate the anti-Sporothrix sp. activity of the essential oil of Origanum majorana Linn. (marjoram), its chemical analysis, and its cytotoxic activity. A total of 18 fungal isolates of Sporothrix brasiliensis (n: 17) from humans, dogs and cats, and a standard strain of Sporothrix schenckii (n: 1) were tested using the broth microdilution technique (Clinical and Laboratory Standard Institute - CLSI M27-A3) and the results were expressed in minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). The MIC50 and MIC90 of itraconazole against S. brasiliensis were 2μg/mL and 8μg/mL, respectively, and the MFC50 and MFC90 were 2μg/mL and >16μg/mL, respectively, with three S. brasiliensis isolates resistant to antifungal. S. schenckii was sensitive at MIC of 1μg/mL and MFC of 8μg/mL. For the oil of O. majorana L., all isolates were susceptible to MIC of ≤2.25-9mg/mL and MFC of ≤2.25-18mg/mL. The MIC50 and MIC90 were ≤2.25mg/mL and 4.5mg/mL, respectively, and the MFC50/90 values were twice more than the MIC. Twenty-two compounds were identified by gas chromatography with a flame ionization detector (CG-FID) and 1,8-cineole and 4-terpineol were the majority. Through the colorimetric (MTT) assay, the toxicity was observed in 70-80% of VERO cells between 0.078 and 5mg/mL. For the first time, the study demonstrated the satisfactory in vitro anti-Sporothrix sp. activity of marjoram oil and further studies are needed to ensure its safe and effective use.
Dai, Longjun; Kang, Guijuan; Li, Yu; Nie, Zhiyi; Duan, Cuifang; Zeng, Rizhong
The rubber particle is a special organelle in which natural rubber is synthesised and stored in the laticifers of Hevea brasiliensis. To better understand the biological functions of rubber particles and to identify the candidate rubber biosynthesis-related proteins, a comprehensive proteome analysis was performed on H. brasiliensis rubber particles using shotgun tandem mass spectrometry profiling approaches-resulting in a thorough report on the rubber particle proteins. A total of 186 rubber particle proteins were identified, with a range in relative molecular mass of 3.9-194.2 kDa and in isoelectric point values of 4.0-11.2. The rubber particle proteins were analysed for gene ontology and could be categorised into eight major groups according to their functions: including rubber biosynthesis, stress- or defence-related responses, protein processing and folding, signal transduction and cellular transport. In addition to well-known rubber biosynthesis-related proteins such as rubber elongation factor (REF), small rubber particle protein (SRPP) and cis-prenyl transferase (CPT), many proteins were firstly identified to be on the rubber particles, including cyclophilin, phospholipase D, cytochrome P450, small GTP-binding protein, clathrin, eukaryotic translation initiation factor, annexin, ABC transporter, translationally controlled tumour protein, ubiquitin-conjugating enzymes, and several homologues of REF, SRPP and CPT. A procedure of multiple reaction monitoring was established for further protein validation. This comprehensive proteome data of rubber particles would facilitate investigation into molecular mechanisms of biogenesis, self-homeostasis and rubber biosynthesis of the rubber particle, and might serve as valuable biomarkers in molecular breeding studies of H. brasiliensis and other alternative rubber-producing species.
Voigt, Carmen Lúcia; da Silva, Cleber Pinto; Doria, Halina Binde; Randi, Marco Antônio Ferreira; de Oliveira Ribeiro, Ciro Alberto; de Campos, Sandro Xavier
From the concentration in water and sediments, bioconcentration and bioaccumulation of copper (Cu), manganese (Mn), zinc (Zn), iron (Fe), cobalt (Co), cadmium (Cd), chrome (Cr), silver (Ag), lead (Pb), nickel (Ni), aluminum (Al), and arsenic (As) were determined in the gills, liver, and muscles of Geophagus brasiliensis in the Alagados Reservoir, Ponta Grossa, Paraná, Brazil. Metals were quantified through AAS, and a study was carried out on the existing relations between metal and body weight, size, and genre of this species. The level of metal in the water of the reservoir was lower than the maximum set forth in the legislation, except for that of Cd and Fe. In sediments, Cu, Cd, Cr, and Ni presented concentrations above the threshold effect level (TEL). Pb and Cr were above the limits for the G. brasiliensis. The tendency of metals present in the muscles of G. brasiliensis was Al > Cu > Zn > Fe > Co > Mn > Cr > Ag > Ni > Pb > Cd > As. In the gills, it was Al > Fe > Zn > Mn > Co > Ag > Cr > Ni > Cu > As > Pb > Cd, and the liver presented Al > Cu > Zn > Co > Fe > Mn > Pb > Ag > Ni > Cr > As > Cd. The bioconcentration and bioaccumulation of metal in the tissues follow the global tendency liver > gills > muscle. The statistical analysis did not point to significant differences in the metal concentration and body weight, size, and gender of the species in the three tissues under analysis.
Marchant, Axelle; Mougel, Florence; Jacquin-Joly, Emmanuelle; Costa, Jane; Almeida, Carlos Eduardo; Harry, Myriam
Background In Latin America, the bloodsucking bugs Triatominae are vectors of Trypanosoma cruzi, the parasite that causes Chagas disease. Chemical elimination programs have been launched to control Chagas disease vectors. However, the disease persists because native vectors from sylvatic habitats are able to (re)colonize houses—a process called domiciliation. Triatoma brasiliensis is one example. Because the chemosensory system allows insects to interact with their environment and plays a key role in insect adaption, we conducted a descriptive and comparative study of the chemosensory transcriptome of T. brasiliensis samples from different ecotopes. Methodology/Principal Finding In a reference transcriptome built using de novo assembly, we found transcripts encoding 27 odorant-binding proteins (OBPs), 17 chemosensory proteins (CSPs), 3 odorant receptors (ORs), 5 transient receptor potential channel (TRPs), 1 sensory neuron membrane protein (SNMPs), 25 takeout proteins, 72 cytochrome P450s, 5 gluthatione S-transferases, and 49 cuticular proteins. Using protein phylogenies, we showed that most of the OBPs and CSPs for T. brasiliensis had well supported orthologs in the kissing bug Rhodnius prolixus. We also showed a higher number of these genes within the bloodsucking bugs and more generally within all Hemipterans compared to the other species in the super-order Paraneoptera. Using both DESeq2 and EdgeR software, we performed differential expression analyses between samples of T. brasiliensis, taking into account their environment (sylvatic, peridomiciliary and domiciliary) and sex. We also searched clusters of co-expressed contigs using HTSCluster. Among differentially expressed (DE) contigs, most were under-expressed in the chemosensory organs of the domiciliary bugs compared to the other samples and in females compared to males. We clearly identified DE genes that play a role in the chemosensory system. Conclusion/Significance Chemosensory genes could be good
Trindade-Silva, Amaro E.; Rua, Cintia P. J.; Andrade, Bruno G. N.; Vicente, Ana Carolina Paulo; Silva, Genivaldo G. Z.
Microbes associated with marine sponges are considered important producers of bioactive, structurally unique polyketides. The synthesis of such secondary metabolites involves type I polyketide synthases (PKSs), which are enzymes that reach a maximum complexity degree in bacteria. The Haplosclerida sponge Arenosclera brasiliensis hosts a complex microbiota and is the source of arenosclerins, alkaloids with cytotoxic and antibacterial activity. In the present investigation, we performed high-throughput sequencing of the ketosynthase (KS) amplicon to investigate the diversity of PKS genes present in the metagenome of A. brasiliensis. Almost 4,000 ketosynthase reads were recovered, with about 90% annotated automatically as bacterial. A total of 235 bacterial KS contigs was rigorously assembled from this sequence pool and submitted to phylogenetic analysis. A great diversity of six type I PKS groups has been consistently detected in our phylogenetic reconstructions, including a novel and A. brasiliensis-exclusive group. Our study is the first to reveal the diversity of type I PKS genes in A. brasiliensis as well as the potential of its microbiome to serve as a source of new polyketides. PMID:23275501
This work reports a systematic comparative study of the properties of natural lattices and rubbers extracted from Hancornia speciosa Gomes and Hevea brasiliensis [(Willd. ex Adr. de Juss.) Muell.-Arg.] (clone RRIM 600) trees from 11 collections in Brazil throughout 2004. Natural rubber latex particl...
Oliveira, Juliana Velasco de Castro; Borges, Thuanny A; Corrêa dos Santos, Renato Augusto; Freitas, Larissa F D; Rosa, Carlos Augusto; Goldman, Gustavo Henrique; Riaño-Pachón, Diego Mauricio
A novel ustilaginomycetous yeast isolated from the intestinal tract of an insect pest of sugarcane roots in Ribeirão Preto, São Paulo State, Brazil, represents a novel species of the genus Pseudozyma based on molecular analyses of the D1/D2 rDNA large subunit and the internal transcribed spacer (ITS1+ITS2) regions. The name Pseudozyma brasiliensis sp. nov. is proposed for this species, with GHG001(T) ( = CBS 13268(T) = UFMG-CM-Y307(T)) as the type strain. P. brasiliensis sp. nov. is a sister species of Pseudozyma vetiver, originally isolated from leaves of vetiver grass and sugarcane in Thailand. P. brasiliensis sp. nov. is able to grow well with xylan as the sole carbon source and produces high levels of an endo-1,4-xylanase that has a higher specific activity in comparison with other eukaryotic xylanases. This enzyme has a variety of industrial applications, indicating the great biotechnological potential of P. brasiliensis.
Mendonça, Vagner José; Alevi, Kaio Cesar Chaboli; Pinotti, Heloisa; Gurgel-Gonçalves, Rodrigo; Pita, Sebastián; Guerra, Ana Letícia; Panzera, Francisco; De Araújo, Renato Freitas; Azeredo-Oliveir, Maria Tercília Vilela De; Rosa, João Aristeu Da
Triatoma bahiensis Sherlock & Serafim, 1967, T. lenti Sherlock & Serafim, 1967, and T. pessoai Sherlock & Serafim, 1967 were described based on material collected in the Brazilian state of Bahia. These species were later included in the T. brasiliensis complex based on their geographic distribution. Triatoma bahiensis and T. pessoai were subsequently synonymized with T. lenti. However, the phylogenetic position of T. lenti within the T. brasiliensis complex has remained doubtful. This study aims to assess the taxonomic status of T. bahiensis and to infer the phylogenetic relationships between T. lenti, T. bahiensis and the other members of the T. brasiliensis species complex. The identities of the species in concern were confirmed by comparisons with high resolution photos of the respective type materials; lectotypes are designated for T. pessoai and T. bahiensis. Morphological, morphometric, molecular, and cytogenetic approaches as well as experimental crosses were used. The low viability of experimental crosses combined with morphological and morphometric data allow the differentiation of T. bahiensis and T. lenti. Pairwise cyt b sequence divergence between T. lenti and T. bahiensis was 2.5%. Cytogenetic and molecular analyses grouped T. lenti and T. bahiensis as members of the T. brasiliensis complex. These results revalidate the specific status of T. bahiensis.
Maia, V C; Fleury, G; Soares, G L G; Isaias, R M S
The gall of Pisphondylia brasiliensis on Guapira opposita, its female and pupa are described and illustrated. The geographic distribution of this species is now widened to Minas Gerais and Rio Grande do Sul (Brazil). For the first time, a female of the genus is described.
Volke-Sepulveda, Tania; Salgado-Bautista, Daniel; Bergmann, Carl; Wells, Lance; Gutierrez-Sanchez, Gerardo; Favela-Torres, Ernesto
The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.
Morais, Cássio Resende; Carvalho, Stephan Malfitano; Araujo, Galber Rodrigues; Souto, Henrique Nazareth; Bonetti, Ana Maria; Morelli, Sandra; Campos Júnior, Edimar Olegário
This study used the pearl cichlid Geophagus brasiliensis as a bioindicator to survey the health of the aquatic environment on four sites (P1, P2, P3 and P4) of the Mumbuca stream located at Monte Carmelo/MG, Brazil. The selection of different sites was made with reference to the gradient of urban activity and via physicochemical and biological evaluation of water quality and genotoxicity. The water quality index was classified as 'good' for P1 and P4, regular in P2 and 'poor' for P3. The micronuclei (MN) frequency obtained from blood analysis was in agreement with the water quality, such that the higher values of MN were detected in sites evaluated as poor. Water degradation conditions worsen according to the flow of the stream over the sites P1, P2 and P3, but for site P4, located after the Monte Carmelo Sewage Treatment Plant, improvements in the micronuclei frequency are detected. Our results showed high levels of potentially toxic metals (chromium, lead, aluminum and nickel) in specific stream sites (P2 and P3). We suggest that the micronuclei induction in G. brasiliensis could be due to the presence of these compounds.
Guidelli, Eder José; Ramos, Ana Paula; Zaniquelli, Maria Elisabete D.; Baffa, Oswaldo
Colloidal silver nanoparticles were synthesized by an easy green method using thermal treatment of aqueous solutions of silver nitrate and natural rubber latex (NRL) extracted from Hevea brasiliensis. The UV-Vis spectra detected the characteristic surface plasmonic absorption band around 435 nm. Both NRL and AgNO 3 contents in the reaction medium have influence in the Ag nanoparticles formation. Lower AgNO 3 concentration led to decreased particle size. The silver nanoparticles presented diameters ranging from 2 nm to 100 nm and had spherical shape. The selected area electron diffraction (SAED) patterns indicated that the silver nanoparticles have face centered cubic (fcc) crystalline structure. FTIR spectra suggest that reduction of the silver ions are facilitated by their interaction with the amine groups from ammonia, which is used for conservation of the NRL, whereas the stability of the particles results from cis-isoprene binding onto the surface of nanoparticles. Therefore natural rubber latex extracted from H. brasiliensis can be employed in the preparation of stable aqueous dispersions of silver nanoparticles acting as a dispersing and/or capping agent. Moreover, this work provides a new method for the synthesis of silver nanoparticles that is simple, easy to perform, pollutant free and inexpensive.
Guidelli, Eder José; Ramos, Ana Paula; Zaniquelli, Maria Elisabete D; Baffa, Oswaldo
Colloidal silver nanoparticles were synthesized by an easy green method using thermal treatment of aqueous solutions of silver nitrate and natural rubber latex (NRL) extracted from Hevea brasiliensis. The UV-Vis spectra detected the characteristic surface plasmonic absorption band around 435 nm. Both NRL and AgNO(3) contents in the reaction medium have influence in the Ag nanoparticles formation. Lower AgNO(3) concentration led to decreased particle size. The silver nanoparticles presented diameters ranging from 2 nm to 100 nm and had spherical shape. The selected area electron diffraction (SAED) patterns indicated that the silver nanoparticles have face centered cubic (fcc) crystalline structure. FTIR spectra suggest that reduction of the silver ions are facilitated by their interaction with the amine groups from ammonia, which is used for conservation of the NRL, whereas the stability of the particles results from cis-isoprene binding onto the surface of nanoparticles. Therefore natural rubber latex extracted from H. brasiliensis can be employed in the preparation of stable aqueous dispersions of silver nanoparticles acting as a dispersing and/or capping agent. Moreover, this work provides a new method for the synthesis of silver nanoparticles that is simple, easy to perform, pollutant free and inexpensive.
The Leydig cells of the bat Tadarida brasiliensis, exhibit two well-defined periods of secretory activity that are intimately associated to the bat reproductive cycle. During the breeding season (August-September, late Winter and early Spring in the southern hemisphere), the interstitial tissue contains hypertrophic Leydig cells characterized ultrastructurally by the presence of pleomorphic mitochondria, depletion of lipid droplets, proliferation of membranes of agranular endoplasmic reticulum (AER) and enlargement of the Golgi complexes. By contrast, from Spring to Fall concurrent with regression of seminiferous tubules, the Leydig cells acquire a quiescent appearance with reduction in size and volume of AER membranes, atrophy of the Golgi complex and a massive storage of lipid droplets. The changes occurring in Leydig cells during the breeding season can be duplicated experimentally in non-breeding bats with exogenous stimulation with hCG. The gonadotropic treatment induces rapid changes in both interstitial cells and seminiferous tubules. The latter present evident signs of reactivation including proliferation of the spermatogenic cell line, permeation of the tubular lumen and depletion of lipid droplets. The Leydig cells display similar features to those found in the bat during the mating season at the peak of secretory activity. The bat T. brasiliensis is an excellent model to correlate the morphological organization of the Leydig cells with either seasonal fluctuations of its secretory activity or after experimental stimulation with gonadotropins.
Méndez-Tovar, Luis J; Mondragón-González, Rafael; Vega-López, Francisco; Dockrell, Hazel M; Hay, Roderick; López-Martínez, Rubén; Manzano-Gayosso, Patricia; Hernández-Hernández, Francisca; Padilla-Desgarennes, Carmen; Bonifaz, Alexandro
IFN-gamma, TNF-alpha, IL-4, IL-10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensis and in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensis crude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosis were used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-gamma production was higher in the healthy controls than in the patients, whereas TNF-alpha secretion was slightly higher in the patients' cultures. IL-4 was detected in the patients' cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-gamma production, and higher concentrations of IL-4, IL-10 and TNF-alpha in the patients' PBMC cultures, indicating that they probably have a Th2 type of immune response.
Leite, J R; Freitas, M O; Sanches, E G; Gomes, M L M; Hostim-Silva, M; Cole, K S
Many aspects of sex change in reef fishes have been studied, including behavior and social organization. However, gonad histology remains the most robust way to identify sexual patterns in fishes. Some uncommon tissues remain poorly described, such as the accessory gonadal structures found in species from the Gobiidae family, which are rare in other bony fishes. This is the first report of the testicular gland in Gramma brasiliensis and for the Grammatidae family. Between April 2011 and February 2012 eighty specimens were collected during four dive campaigns on the Taipus de Fora reef (13°56'20"S 38°55'32"W), Bahia, Northeast Brazil, and their sex was determined. Thirteen per cent of the active-females and 90% of the active-males had testicular gland tissue in their ovotestis. This discovery led to additional research into the characteristics of the gland tissue and its relationship with gonadal maturation. Three patterns of testicular gland development were found in Brazilian basslet ovotestis. Both ova and sperm-producing gonad contained testicular gland tissue, and the appearance of this tissue seems to be the first modification of ovotestis tissue marking the beginning of the protogynous sex-change process in G. brasiliensis.
Cultivated strains of Agaricus bisporus and A. brasiliensis: chemical characterization and evaluation of antioxidant and antimicrobial properties for the final healthy product--natural preservatives in yoghurt.
Stojković, Dejan; Reis, Filipa S; Glamočlija, Jasmina; Ćirić, Ana; Barros, Lillian; Van Griensven, Leo J L D; Ferreira, Isabel C F R; Soković, Marina
Agaricus bisporus (J. E. Lange) Emil J. Imbach and Agaricus brasiliensis Wasser, M. Didukh, Amazonas & Stamets are edible mushrooms. We chemically characterized these mushrooms for nutritional value, hydrophilic and lipophilic compounds. The antioxidant and antimicrobial activities of methanolic and ethanolic extracts were assessed. Hepatotoxicity was also evaluated. The ethanolic extract of both species was tested for inhibition of Listeria monocytogenes growth in yoghurt. Both species proved to be a good source of bioactive compounds. A. brasiliensis was richer in polyunsaturated fatty acids and revealed the highest concentration of phenolic acids, and tocopherols. A. bisporus showed the highest monounsaturated fatty acids and ergosterol contents. A. brasiliensis revealed the highest antioxidant potential, and its ethanolic extract displayed the highest antibacterial potential; the methanolic extract of A. bisporus revealed the highest antifungal activity. A. brasiliensis possessed better preserving properties in yoghurt.
Turmelle, Amy S; Allen, Louise C; Jackson, Felix R; Kunz, Thomas H; Rupprecht, Charles E; McCracken, Gary F
Previous studies have investigated rabies virus (RABV) epizootiology in Brazilian free-tailed bats (Tadarida brasiliensis) in natural cave roosts. However, little is known about geographic variation in RABV exposure, or if the use of man-made roosts by this species affects enzootic RABV infection dynamics within colonies. We sampled rabies viral neutralizing antibodies in bats at three bridge and three cave roosts at multiple time points during the reproductive season to investigate temporal and roost variation in RABV exposure. We report seropositive bats in all age and sex classes with minimal geographic variation in RABV seroprevalence among Brazilian free-tailed bat colonies in south-central Texas. While roost type was not a significant predictor of RABV seroprevalence, it was significantly associated with seasonal fluctuations, suggesting patterns of exposure that differ between roosts. Temporal patterns suggest increased RABV seroprevalence after parturition in cave colonies, potentially related to an influx of susceptible young, in contrast to more uniform seroprevalence in bridge colonies. This study highlights the importance of life history and roost ecology in understanding patterns of RABV seroprevalence in colonies of the Brazilian free-tailed bat.
Guerra, A L; Alevi, K C C; Banho, C A; Oliveira, J; Rosa, J A; Azeredo-Oliveira, M T V
The Brasiliensis subcomplex is a monophyletic group formed by the species Triatoma brasiliensis brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki. However, using cytogenetic data and experimental hybrid crosses, T. lenti and T. petrochiae were also grouped into this subcomplex. This study aims to analyze the properties of hotspot in the D2 domain of the nuclear gene 28S in all species of the Brasiliensis subcomplex as well as T. lenti and T. petrochiae. These species show two transversions at position 385 (G↔C and T↔G). We suggest that this mutation in haplotype 4 may be an initial molecular tool that supports the relationship of these species with the subcomplex. In addition to the transversion at haplotype 4, these species, aside from T. melanica, also possess a transversion at position 385 (G↔T) in haplotype 1. Thus, we describe the hotspot mutations of the D2 domain of the nuclear gene 28S for species in Brasiliensis subcomplex as follows: three transversions are present at position 385 of haplotypes 1 and 4, which are shared by members of the subcomplex as well as T. lenti and T. petrochiae. These transversions may be considered a synapomorphy between these species. However, we emphasize that new phylogenetic studies should be conducted to evaluate whether T. lenti and T. petrochiae are truly members of the Brasiliensis subcomplex.
Taborda, Paulo R.; Taborda, Valeria A.; McGinnis, Michael R.
The new genus Lacazia P. Taborda, V. Taborda, et McGinnis is proposed to accommodate Lacazia loboi (O. M. Fonseca et Lacaz) P. Taborda, V. Taborda, et McGinnis, the obligate pathogen that causes lobomycosis in mammals. The continued placement of that fungus in the genus Paracoccidioides Almeida as Paracoccidioides loboi is taxonomically inappropriate. Loboa loboi Ciferri et al. is a synonym of Paracoccidioides brasiliensis. PMID:10325371
Nembo, B; Goudey-Perriere, F; Gayral, P; Perriere, C; Brousse-Gaury, P
Microtopographic features of the various growth stages of the three free-living larval stages of the rat hookworm Nippostrongylus brasiliensis (Nematoda) were surveyed by scanning electron microscopy. These worms have a rounded anterior end and an elongated tail. Cuticular annulations were observed along the body, which also bore two ribbon-like lateral alae. Two rings of six lip-like lappets were observed around the triradiate oral opening in all larval stages. The cephalic space contained two lateral amphidial pits. The excretory pore in the third anterior part was observed in a ventral view of the larvae. No deirids were observed. The anus with a crescent-shape opening was located posteriorly. Phasmidial apertures, only observed in the third-stage larvae, opened on the lateral alae in the tail region.
Flores, Gema; Dastmalchi, Keyvan; Paulino, Sturlainny; Whalen, Kathleen; Dabo, Abdoulaye J; Reynertson, Kurt A; Foronjy, Robert F; D'Armiento, Jeanine M; Kennelly, Edward J
Nine anthocyanins (1-9) from the edible fruits of Eugenia brasiliensis were identified by HPLC-PDA and LC-MS, and seven of these are described for the first time in this Brazilian fruit. Two of the major anthocyanins, delphinidin (8) and cyanidin (9), were studied for their inhibitory activity against chemokine interleukin-8 (IL-8) production before and after cigarette smoke extract (CSE) treatment of cells. In non-treated cells the amount of IL-8 was unchanged following treatment with cyanidin and delphinidin in concentrations 0.1-10 μM. Both delphinidin (8) and cyanidin (9) decreased the production of IL-8 in treated cells, at 1 and 10 μM, respectively. Delphinidin (8) demonstrated IL-8 inhibition in the CSE treated cells in a dose-dependent manner.
Flores, Gema; Dastmalchi, Keyvan; Paulino, Sturlainny; Whalen, Kathleen; Dabo, Abdoulaye J.; Reynertson, Kurt A.; Foronjy, Robert F.; D Armiento, Jeanine M.; Kennelly, Edward J.
Nine anthocyanins (1–9) from the edible fruits of Eugenia brasiliensis were identified by HPLC-PDA and LC-MS, and seven of these are described for the first time in this Brazilian fruit. Two of the major anthocyanins, delphinidin (8) and cyanidin (9), were studied for their inhibitory activity against chemokine interleukin-8 (IL-8) production before and after cigarette smoke extract (CSE) treatment of cells. In non-treated cells the amount of IL-8 was unchanged following treatment with cyanidin and delphinidin in concentrations 0.1–10 M. Both delphinidin (8) and cyanidin (9) decreased the production of IL-8 in treated cells, at 1 M and 10 M, respectively. Delphinidin (8) demonstrated IL-8 inhibition in the CSE treated cells in a dose-dependent manner. PMID:25005941
Bagstad, Kenneth J.; Widerholdt, Ruscena
Migratory species provide diverse ecosystem services to people, but these values have seldom been estimated rangewide for a single species. In this article, we summarize visitation and consumer surplus for recreational visitors to viewing sites for the Mexican free-tailed bat (Tadarida brasiliensis mexicana) throughout the Southwestern United States. Public bat viewing opportunities are available at 17 of 25 major roosts across six states; on an annual basis, we estimate that over 242,000 visitors view bats, gaining over $6.5 million in consumer surplus. A better understanding of spatial mismatches between the areas where bats provide value to people and areas most critical for maintaining migratory populations can better inform conservation planning, including economic incentive systems for conservation.
Canals, Mauricio; Sabat, Pablo; Veloso, Claudio
The bronchial tree of most mammalian lungs is a good example of an efficient distribution system whose geometry and dimensions of branched structures are important factors in determining the efficiency of respiration. Small and flying endothermic animals have high-energy requirements, requiring morphological and physiological adaptations to reduce energy loss. Here we show that Tadarida brasiliensis, a nocturnal small bat whose energy requirements are exacerbated by this small size and by their frequent exposure to high altitude, has a different morphology in the proximal airway, sustained by a wider trachea and better scaling factors, than other non-flying mammals. This design allows a great decrease of the volume specific resistance of the proximal airway and in consequence a very low entropy production during breathing, approximately 1/18 of that expected for a non-flying mammals of similar body size.
Moravec, F; Santos, C P
Dracunculus brasiliensis sp. n. (Dracunculidae), is described based on a single female specimen found in the body cavity of the anaconda, Eunectes murinus (L.) (Ophidia: Boidae), from the Mexiana Island, Amazon River delta, Brazil and one female previously recorded from the subcutaneous tissue of this host species imported from South America into Europe (ZOO in the Czech Republic). The new species is characterised mainly by markedly large, anteriorly protruding dorsal and ventral double papillae of the internal circle and small lateral papillae of the same circle, a widely rounded caudal end, the excretory pore situated just posterior to the nerve ring, a distinctly transversely striated cuticle and by the length (396-429 mum) of larvae from uterus. This is the first species of Dracunculus described from reptiles in South America.
Seyedmousavi, S; Guillot, J; Tolooe, A; Verweij, P E; de Hoog, G S
Zoonotic fungi can be naturally transmitted between animals and humans, and in some cases cause significant public health problems. A number of mycoses associated with zoonotic transmission are among the group of the most common fungal diseases, worldwide. It is, however, notable that some fungal diseases with zoonotic potential have lacked adequate attention in international public health efforts, leading to insufficient attention on their preventive strategies. This review aims to highlight some mycoses whose zoonotic potential received less attention, including infections caused by Talaromyces (Penicillium) marneffei, Lacazia loboi, Emmonsia spp., Basidiobolus ranarum, Conidiobolus spp. and Paracoccidioides brasiliensis.
Abreu e Silva, Mariana Àlvares de; Salum, Fernanda Gonçalves; Figueiredo, Maria Antonia; Cherubini, Karen
Paracoccidioidomycosis is a deep mycosis endemic to Latin America, with considerable morbidity and mortality. It is caused by the dimorphic fungus Paracoccidioides brasiliensis, which affects, among other organs in the human body, the oral cavity. Fungus virulence and immunocompetence of the host determine the establishment of infection or active disease, whose severity and clinical behaviour depend mostly on the cellular immune response of the host. Often, oral lesions constitute the first sign and site of confirmation of diagnosis, which in most cases is delayed. The success of the treatment depends on early and correct diagnosis, as well as on the patient's adherence to the drug therapy.
Dusotoit-Coucaud, Anaïs; Brunel, Nicole; Kongsawadworakul, Panida; Viboonjun, Unchera; Lacointe, André; Julien, Jean-Louis; Chrestin, Hervé; Sakr, Soulaïman
Background and Aims The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel®) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described. Methods Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel. Key Results Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application. Conclusion The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees. PMID:19567416
Matsumoto-Akanuma, Akiko; Akanuma, Satoshi; Motoi, Masuro; Yamagishi, Akihiko; Ohno, Naohito
Laccase isozymes have been identified in several fungi. We report the cloning of 4 laccase genes from the medicinal mushroom Agaricus brasiliensis. The lac1 gene contained a 1560-base pair (bp) open reading frame (ORF) encoding 520 amino acids that was interrupted with 14 introns in genomic DNA. The deduced amino acid sequence indicated a multicopper oxidase signature 1 and 2 multicopper oxidase signature 2. The lac2 gene contained a 1566-bp ORF encoding 522 amino acids that was interrupted with 13 introns in genomic DNA. A number of different nucleotides were observed in whole regions containing the substitution of amino acid residues (lac2a and lac2b). The partial DNA fragments of lac3 and lac4 genes were subcloned using the semi-random two-step polymerase chain reaction method. The lac3 and lac4 genes contained coding sequences with a 1575-bp ORF encoding 525 amino acids and a 1584-bp ORF encoding 528 amino acids, respectively. However, the whole complementary DNA fragment of both laccases could not be amplified with polymerase chain reaction against the complementary DNA library; therefore, introns were deduced based on the GT-AG rule and multiple alignment of laccases from other fungi, which showed high identity. All laccases from A. brasiliensis conserved the fungal laccase signature sequence and suggest 2 subfamilies according to the location of introns and phylogenetic analysis. The genes lac2 and lac4 had a high degree of identity, and the lac2a gene was located upstream of the lac4 gene.
Background Human activities, such as agriculture, hunting, and habitat modification, exert a significant effect on native species. Although many species have suffered population declines, increased population fragmentation, or even extinction in connection with these human impacts, others seem to have benefitted from human modification of their habitat. Here we examine whether population growth in an insectivorous bat (Tadarida brasiliensis mexicana) can be attributed to the widespread expansion of agriculture in North America following European settlement. Colonies of T. b. mexicana are extremely large (~106 individuals) and, in the modern era, major agricultural insect pests form an important component of their food resource. It is thus hypothesized that the growth of these insectivorous bat populations was coupled to the expansion of agricultural land use in North America over the last few centuries. Results We sequenced one haploid and one autosomal locus to determine the rate and time of onset of population growth in T. b. mexicana. Using an approximate Maximum Likelihood method, we have determined that T. b. mexicana populations began to grow ~220 kya from a relatively small ancestral effective population size before reaching the large effective population size observed today. Conclusions Our analyses reject the hypothesis that T. b. mexicana populations grew in connection with the expansion of human agriculture in North America, and instead suggest that this growth commenced long before the arrival of humans. As T. brasiliensis is a subtropical species, we hypothesize that the observed signals of population growth may instead reflect range expansions of ancestral bat populations from southern glacial refugia during the tail end of the Pleistocene. PMID:21457563
Ferguson, A; Logan, R F; MacDonald, T T
We have examined the effects of parasite infection on the mucosal architecture of mice maintained on an elemental diet (Vivonex). Techniques used were conventional histology, micro-dissection and measurement of individual villi and crypts, and measurement of crypt cell proliferation rate by a metaphase accumulation technique. In normal, non-parasitised mice the elemental diet caused no change in villus height, crypt depth, or crypt cell proliferation. Likewise, the only effects of chronic protozoal infection or Nippostrongylus brasiliensis infection on the intestine of mice fed a normal diet have been a slight crypt hypertrophy and an increase in crypt cell proliferation rate without villous atrophy. However, the combination of elemental diet and parasite infection resulted in increased mucosal damage when compared with infected mice on a normal diet. Elemental diet mice infected with the nematode Nippostrongylus brasiliensis had significantly reduced villus height and correspondingly raised crypt length and metaphase accumulation rate. Elemental diet mice infected with the protozoan Giardia muris did not have villous atrophy but there was a significant increase in crypt length and metaphase accumulation when compared with infected normal diet mice. These experiments show that in two animal models of enteric infection, elemental diet has altered the host parasite relationship to the detriment of the host. Images Fig. 1 Fig. 5 PMID:7364318
Characterization of blood beta-1,3-glucan and anti-beta-glucan antibody in hemodialysis patients using culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).
Ishibashi, Ken-ichi; Yoshida, Masaharu; Nakabayashi, Iwao; Yoshikawa, Noriko; Miura, Noriko N; Adachi, Yoshiyuki; Ohno, Naohito
Beta-glucan is a major component of fungal cell walls and shows various immunopharmacological activities including antitumor activity. Previously, we detected anti-beta-glucan antibody in human sera. Anti-beta-glucan antibody participates in the immune response to fungal cell wall beta-glucan. Patients on dialysis are at high risk of infection including fungal infections. We examined the plasma beta-glucan level and the titer of anti-beta-glucan antibody in dialysis patients. We measured plasma beta-1,3-glucan concentrations with the limulus G test and anti-beta-glucan antibody titers by ELISA with Candida beta-glucan-coated plates. We also examined the influence of the period of dialysis and the kind of dialysis membrane. The patients were positive for beta-1,3-glucan in their plasma. The anti-beta-glucan antibody titer was lower in the dialysis patients than in healthy volunteers. Long-term dialysis patients showed lower anti-beta-glucan antibody titers than short-term dialysis patients. No significant difference was found between the kinds of dialysis membrane. The titer of anti-beta-glucan antibody as recognition molecule of beta-glucan was low in dialysis patients compared with healthy volunteers. This is likely to be one factor explaining the sensitivity to infection of the dialysis patients. An appropriate application of culinary-medicinal mushroom such as Agaricus brasiliensis has potential for the prevention of fungal infection in dialysis patients.
Ogawa, Hiroyuki; Matsuoka, Hideaki; Saito, Mikako
The growth of black mold (Aspergillus brasiliensis) in black-colored samples such as hair color and mascara was measured with an automatic count system based on time-lapse shadow image analysis (TSIA). A. brasiliensis suspended in a lecithin and polysorbate (LP) solution of each sample (hair color or mascara) was spread on a potato dextrose agar medium plate containing LP. The background image darkness of the agar plate could be adjusted to attain accurate colony counts. 95 colonies in hair color and 22 colonies in mascara could be automatically determined at 48 h. The accuracy of the colony counts could be confirmed from the timelapse image data. In contrast, conventional visual counting at a specified time could not determine the number of colonies or led to false colony counts.
Vera-Cabrera, Lucio; Johnson, Wendy M.; Welsh, Oliverio; Resendiz-Uresti, Francisco L.; Salinas-Carmona, Mario C.
An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3′-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria. PMID:10325357
Background The rubber tree, Hevea brasiliensis, is a species native to the Brazilian Amazon region and it supplies almost all the world’s natural rubber, a strategic raw material for a variety of products. One of the major challenges for developing rubber tree plantations is adapting the plant to biotic and abiotic stress. Transcriptome analysis is one of the main approaches for identifying the complete set of active genes in a cell or tissue for a specific developmental stage or physiological condition. Results Here, we report on the sequencing, assembling, annotation and screening for molecular markers from a pool of H. brasiliensis tissues. A total of 17,166 contigs were successfully annotated. Then, 2,191 Single Nucleotide Variation (SNV) and 1.397 Simple Sequence Repeat (SSR) loci were discriminated from the sequences. From 306 putative, mainly non-synonymous SNVs located in CDS sequences, 191 were checked for their ability to characterize 23 Hevea genotypes by an allele-specific amplification technology. For 172 (90%), the nucleotide variation at the predicted genomic location was confirmed, thus validating the different steps from sequencing to the in silico detection of the SNVs. Conclusions This is the first study of the H. brasiliensis transcriptome, covering a wide range of tissues and organs, leading to the production of the first developed SNP markers. This process could be amplified to a larger set of in silico detected SNVs in expressed genes in order to increase the marker density in available and future genetic maps. The results obtained in this study will contribute to the H. brasiliensis genetic breeding program focused on improving of disease resistance and latex yield. PMID:24670056
Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Peruch, Frédéric
This review article aims to gather all the knowledge on two important proteins associated with Hevea brasiliensis rubber particles: namely the rubber elongation factor (REF) and the small rubber particle protein (SRPP). It covers more then three decades of research on these two proteins and their homologues in plants, and particularly emphasizes on the different possible properties or functions of these various proteins found in plants.
Borba-Santos, Luana Pereira; Gagini, Thalita; Ishida, Kelly; de Souza, Wanderley; Rozental, Sonia
Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1-2 µg ml(-1). Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.
Monteiro, Cristiane Schüler; Miguel, Obdulio G; Eugênia, Balbi Maria; Penteado, Patrícia Teixeira Padilha Da Silva; Haracemiv, Sonia Maria Chaves
Lycopene belongs to the subgroup of non-oxygenated carotenoids with antioxidant and anti-carcinogenic properties that are comparatively more powerful than the majority of plasma carotenoids. When foodstuffs containing lycopene are processed, the cell wall breaks down during the thermal process--thus enabling the extraction of lycopene from chromoplasts, improving their bioavailability. Edible mushroom Agaricus brasiliensis stands out given its medicinal properties and antioxidant potential when used to treat heart diseases and to prevent cancer. Given the interest in lycopene-rich foods, the purpose of the present study was to determine the lycopene present in different types of tomato sauce with A. brasiliensis and/or its extract by high-performance liquid chromatography. The type of solvent (dichloromethane, hexane and ethanol) to remove water from the tomato sauce was tested before the extraction of carotenoids. Lycopene determination in tomato sauces, in tomatoes and in the A. brasiliensis extract was carried out via high-performance liquid chromatography. Findings show that when tomato sauce and raw materials underwent heat treatment, the type of treatment did not interfere with carotenoid and lycopene bioavailability--indicating that those sauces have a significant concentration of carotenoids and, in particular, their content in the lycopene proportion compared with total carotenoids.
Eller, Cleiton B; Lima, Aline L; Oliveira, Rafael S
Foliar water uptake (FWU) is a common water acquisition mechanism for plants inhabiting temperate fog-affected ecosystems, but the prevalence and consequences of this process for the water and carbon balance of tropical cloud forest species are unknown. We performed a series of experiments under field and glasshouse conditions using a combination of methods (sap flow, fluorescent apoplastic tracers and stable isotopes) to trace fog water movement from foliage to belowground components of Drimys brasiliensis. In addition, we measured leaf water potential, leaf gas exchange, leaf water repellency and growth of plants under contrasting soil water availabilities and fog exposure in glasshouse experiments to evaluate FWU effects on the water and carbon balance of D. brasiliensis saplings. Fog water diffused directly through leaf cuticles and contributed up to 42% of total foliar water content. FWU caused reversals in sap flow in stems and roots of up to 26% of daily maximum transpiration. Fog water transported through the xylem reached belowground pools and enhanced leaf water potential, photosynthesis, stomatal conductance and growth relative to plants sheltered from fog. Foliar uptake of fog water is an important water acquisition mechanism that can mitigate the deleterious effects of soil water deficits for D. brasiliensis.
Pereira, R T; Costa, L S; Oliveira, I R C; Araújo, J C; Aerts, M; Vigliano, F A; Rosa, P V
The endocrine cells (ECs) of the gastrointestinal mucosa form the largest endocrine system in the body, not only in terms of cell numbers but also in terms of the different produced substances. Data describing the association between the relative distributions of the peptide-specific ECs in relation to feeding habits can be useful tools that enable the creation of a general expected pattern of EC distribution. We aimed to investigate the distribution of ECs immunoreactive for the peptides gastrin (GAS), cholecystokinin (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in different segments of the digestive tract of carnivorous fish dorado (Salminus brasiliensis) by using immunohistochemistry procedures. The distribution of endocrine cells immunoreactive for gastrin (GAS), cholecystokinin (CCK-8), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) in digestive tract of dorado S. brasiliensis was examined by immunohistochemistry. The results describe the association between the distribution of the peptide-specific endocrine cells and feeding habits in different carnivorous fish. The largest number of endocrine cells immunoreactive for GAS, CCK-8, and CGRP were found in the pyloric stomach region and the pyloric caeca. However, NPY-immunoreactive endocrine cells were markedly restricted to the midgut. The distribution pattern of endocrine cells identified in S. brasiliensis is similar to that found in other carnivorous fishes.
Schwartz, Christine; Tressler, Jedidiah; Keller, Halli; Vanzant, Marc; Ezell, Sarah; Smotherman, Michael
Echolocating insectivorous bats consummate prey captures using a distinct vocal motor pattern commonly known as the terminal or feeding buzz, which is widely considered a fixed motor pattern executed independently of auditory feedback influences. The Mexican free-tailed bat, Tadarida brasiliensis, offers an opportunity to explore the role of sensory feedback in buzzing because they emit similar buzzes both in flight during foraging and while stationary as communication sounds. Here we compared the spectral and temporal patterns of foraging and communication buzzes to address whether or not auditory feedback may influence buzz patterns. We found that while foraging buzzes uttered in open space were composed of generic FM calls, communication buzzes were composed of an adapted CF-FM call similar to the call type used by T. brasiliensis when navigating in confined spaces. This provides the first evidence that some bats can make significant context-dependent changes in the spectral parameters of calls within their buzz. We also found that inter-pulse intervals, but not call durations, were different within the two buzz types. These observations indicate that though a common pattern generator hierarchically organizes all buzzes, T. brasiliensis retains a significant capacity to adapt the spectral and temporal patterns of elements within its buzzes.
Allen, Louise C; Turmelle, Amy S; Mendonça, Mary T; Navara, Kristen J; Kunz, Thomas H; McCracken, Gary F
Bats have recently been implicated as reservoirs of important emerging diseases. However, few studies have examined immune responses in bats, and even fewer have evaluated these responses in an ecological context. We examined aspects of both innate and adaptive immune response in adult female Brazilian free-tailed bats (Tadarida brasiliensis) at four maternity roosts (two natural caves and two human-made bridges) in south-central Texas. Immune measurements included in vitro bactericidal ability of whole blood and in vivo T cell mediated response to mitogenic challenge. Bactericidal activity in T. brasiliensis varied with roosting ecology, but appears to be sensitive to colony-level effects. Blood from females living at one cave had significantly lower bactericidal ability than blood from females at three other sites. T cell mediated response in this species was associated with variation in roost ecology, with females from two caves having greater responses than females from two bridges. T cell mediated response and bactericidal activity were negatively correlated with one another within individuals that were tested for both. Variation in immunological response of T. brasiliensis is important for understanding the influence of the environment on the frequency and distribution of immunologically competent individuals and for understanding disease-host dynamics in this and other colonial species.
Tristão, F.S.M.; Rocha, F.A.; Dias, F.C.; Rossi, M.A.; Silva, J.S.
Paracoccidioidomycosis (PCM) is a chronic systemic mycosis caused by the inhalation of the thermally dimorphic fungus Paracoccidioides brasiliensis as well as the recently described P. lutzii. Because the primary infection occurs in the lungs, we investigated the differential involvement of the right and left lungs in experimental P. brasiliensis infection. Lungs were collected from C57BL/6 mice at 70 days after intravenous infection with 1×106 yeast cells of a virulent strain of P. brasiliensis (Pb18). The left lung, which in mice is smaller and has fewer lobes than the right lung, yielded increased fungal recovery associated with a predominant interleukin-4 response and diminished synthesis of interferon-γ and nitric oxide compared with the right lung. Our data indicate differential involvement of the right and left lungs during experimental PCM. This knowledge emphasizes the need for an accurate, standardized protocol for tissue collection during studies of experimental P. brasiliensis infection, since experiments using the same lungs favor the collection of comparable data among different mice. PMID:24141611
Dos Santos, Carlos Henrique Corrêa; Borges, Izabeau Pontes; da Silva, Virgínia Claudia; de Sousa, Paulo Teixeira; Kawashita, Nair Honda; Baviera, Amanda Martins; Carvalho, Mario Geraldo de
Context Siolmatra brasiliensis (Cogn.) Baill (Cucurbitaceae) is a climbing plant widely used for the treatment of diabetes mellitus symptoms. Objective This work evaluates the antidiabetic activity of an extract of S. brasiliensis in streptozotocin-diabetic rats and promotes the phytochemical investigation to isolate the major compounds of the same extract. Materials and methods Male Wistar rats were divided into normal (N) and diabetic rats (DC) treated with water; diabetic rats treated with 3U insulin (DI) or with 250 (DSb250) or 500 mg/kg (DSb500) of hydroalcoholic extract of the stalks of S. brasiliensis, via oral gavage, for 21 days. Physiological and biochemical parameters classically altered in diabetes were monitored. The triterpenoids were isolated from the ethyl acetate fraction under silica gel column chromatography and Sephadex-LH20 methods and their structures were determined by NMR, HR-ESI-MS and DC analysis. Results When compared with DC, DSb250 rats showed a reduction in the hyperglycemia (DC: 26.46 ± 0.69 versus DSb250: 19.67 ± 1.06 mmol/L) and glycosuria (DC: 43.02 ± 3.19 versus DSb250: 28.46 ± 2.14 mmol/24 h) and increase in hepatic glycogen (DC: 14.44 ± 1.26 versus DSb250: 22.08 ± 4.26 mg/g). Three known cucurbitacins were isolated from a hydroalcoholic extract of S. brasiliensis, i.e., cayaponosides A1, B4, D, and a new dammarane saponin 3-O-β-d-gentiobiosyl-26-O-β-d-glucopyranosyl-20-hydroxydammar-24-ene. The structures of these compounds were elucidated by spectral data analysis of the natural products and their acetyl derivatives. Discussion and conclusion The known cucurbitacins and/or the new identified saponin may be related with the antidiabetic activity of S. brasiliensis.
Sando, Tomoki; Takeno, Shinya; Watanabe, Norie; Okumoto, Hiroshi; Kuzuyama, Tomohisa; Yamashita, Atsushi; Hattori, Masahira; Ogasawara, Naotake; Fukusaki, Eiichiro; Kobayashi, Akio
Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.
Figueiredo, Sônia Aparecida; Vilela, Fernanda Maria Pinto; da Silva, Claudinei Alves; Cunha, Thiago Mattar; Dos Santos, Marcelo Henrique; Fonseca, Maria José Vieira
The damaging effects of sunlight to the skin has triggered studies that involve the synthesis and extraction of organic compounds from natural sources that can absorb UV radiation, and studies on polyphenolic compounds with antioxidant and anti-inflammatory properties that can be used as photochemopreventive agents for reducing skin damage. We investigated the in vitro and in vivo photoprotective/photochemopreventive potential of Garcinia brasiliensis epicarp extract (GbEE). We evaluated the cell viability of L929 fibroblasts after UVB exposure using a quartz plate containing the extract solution or the GbEE formulation. The in vivo photoprotective effect of the GbEE formulation was evaluated by measuring the UVB damage-induced decrease in endogenous reduced glutathione (GSH), the increase in myeloperoxidase (MPO) activity and secretion of cytokines IL-1β and TNF-α. The in vitro methodology using fibroblasts showed that the photoprotective properties of the GbEE solutions and 10% GbEE formulation were similar to the commercial sunscreen (SPF-15). In vivo results demonstrated of the GbEE formulation in decreasing UVB induced-damage such as GSH depletion, an increased in MPO activity and secretion of cytokines IL-1β and TNF-α. The results showed that the extract has great potential for use as a sunscreen in topical formulations in addition to UV filters.
This research aimed to systematically identify and preliminarily validate the Hevea brasiliensis expressed sequence tag (EST) information using Simple Sequence Repeat (SSR) and provide evidence for further development of SSR molecular marker. The definition of general SSR features of Hevea EST splicing sequences and development of SSR primers founded the basis of diversity analysis and variety identification for Hevea tree resource. 1134 SSR loci were identified in the EST splicing sequence and distributed in 840 Unigene. The occurrence rate of SSR loci was 23.9%, and the average distribution distance of EST-SSR was 2.59 kb. The major repeat type was mononucleotide repeat motif, which accounted for 38.89%, while the corresponding value was 36.95% for dinucleotide repeat motif and 18.17% for trinucleotide repeat motif; the proportion of other motifs was only 5.99%. The superior repeat motifs for mononucleotide, dinucleotide, and trinucleotide were A/T, AG/CT, and AAG/CTT, respectively. 739 pair of primers were designed for 1134 SSR loci. PCR amplification was performed on Hevea Reyan5-11, Reyan87-6-47, and PR107, and 180 pairs of primers were selected which were able to amplify polymorphism bands. PMID:28232872
Omar, Atef; Foissner, Wilhelm
Using standard morphological methods, we describe one new Leptopharynx species and a new subspecies of L. costatus, both from soil of the neotropic region. Further, we studied two populations of L. costatus costatus. Leptopharynx brasiliensis nov. spec., which was discovered in the Mato Grosso, Brazil, is a large member (60 μm) of the genus with an enormous oral basket. It differs from similar congeners in having six monokinetids in kinety 6, widely spaced kinetids in kinety 1, and an average of 294 kinetids. Leptopharynx costatus gonohymen nov. subspec., which was discovered in southern Florida, makes a small (35 μm) and a large morph (55 μm) both with narrow oral basket. The small morph is inseparable from the small morph of L. costatus costatus, while the large morph has right-angled adoral membranelles and widely (vs. narrowly) spaced kinetids in kinety 1. The small morphs of a Brazilian and an Austrian L. costatus match Mexican and other European populations, all having on average 181–187 kinetids. As yet, we know four morphs of L. costatus that differ by body size (small vs. large), the oral basket (narrow vs. wide), membranelle 1 (present vs. absent), and the arrangement of the membranelles (flat vs. angled). PMID:21855305
Dai, Longjun; Nie, Zhiyi; Kang, Guijuan; Li, Yu; Zeng, Rizhong
Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).
Shanmugaprakash, M; Vinothkumar, V; Ragupathy, J; Reddy, D Amala
Naringinase is a complex enzyme composed of α-L-rhamnosidase and β-D-glucosidase, which has a vast potential application in the field of industrial biotechnology. The novel aspect in the present study is employing a three-phase partitioning (TPP) technique for the purification of naringinase by solid-state fermentation using Aspergillus brasiliensis MTCC 1344. At optimum conditions of 28±2 °C and 30% (w/v) ammonium sulfate along with a 1:1 ratio of t-butanol to crude extract, the purification is enhanced by 4.2-fold .Temperature and pH profile of TPP purified naringinase was found to be active with an optimal activity of 719.6 units at an elevated temperature of 60 °C. The kinetic constants K(m) and V(max) using naringin as substrate were 3.21 mM and 321 U/ml. The purified enzyme was not inhibited by any metal ions except Hg(2+) but completely inhibited by adding chelating agents such as EDTA and SDS at a concentration of 10 mM. These results can be inevitable to establish the TPP method to be an inexpensive, economical and attractive technology for better recovery and to find its application in the industrial sector.
Miyazaki, Takatsugu; Matsumoto, Yuji; Matsuda, Kana; Kurakata, Yuma; Matsuo, Ichiro; Ito, Yukishige; Nishikawa, Atsushi; Tonozuka, Takashi
A gene for processing α-glucosidase I from a filamentous fungus, Aspergillus brasiliensis (formerly called Aspergillus niger) ATCC 9642 was cloned and fused to a glutathione S-transferase tag. The active construct with the highest production level was a truncation mutant deleting the first 16 residues of the hydrophobic N-terminal domain. This fusion enzyme hydrolyzed pyridylaminated (PA-) oligosaccharides Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA and the products were identified as Glc(2)Man(9)GlcNAc(2)-PA and Glc(2)Man(4)-PA, respectively. Saturation curves were obtained for both Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA, and the K (m) values for both substrates were estimated in the micromolar range. When 1 μM Glc(3)Man(4)-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 μM concentration, both inhibitors reduced activity by 50%.
Grigg, M E; Gounaris, K; Selkirk, M E
Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso-form. The enzyme activity of Ca(2+)-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity of inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase. PMID:8713083
Ji, Weifeng; Huang, Haiying; Chao, Ji; Lu, Wuchao; Guo, Jianyou
Objective. The present investigation examined the neuroprotective effect of Agaricus brasiliensis (AbS) against STZ-induced diabetic neuropathic pain in laboratory rats. STZ-induced diabetic rats were administered orally with AbS. Body weight, serum glucose, and behavioral parameters were measured before and at the end of the experiment to see the effect of AbS on these parameters. After 6 weeks of treatments, all animals were sacrificed to study various biochemical parameters. Treatment with AbS 80 mg/kg in diabetic animals showed significant increase in body weight, pain threshold, and paw withdrawal threshold and significant decrease in serum glucose, LPO and NO level, Na-K-ATPase level, and TNF-α and IL-1β level as compared to vehicle treated diabetic animals in dose and time dependent manner. AbS can offer pain relief in PDN. This may be of potential benefit in clinical practice for the management of diabetic neuropathy. PMID:24527050
O'Shea, Thomas J.
Fat content of migratory Tadarida brasiliensis was determined during the spring, summer and fall of 1972 in the Verde Valley of Arizona. Fat indices were highest in March arrivals, generally declined throughout the summer, and were lowest in September. In both 1972 and 1973 bats had arrived at the study area by mid-March. In 1971 bats were last noted at the area in mid-October while in 1972 they had disappeared by late September. On the basis on physiological calculations it is estimated that bats collected in March 1972 possessed sufficient fat reserves to carry them a mean distance of 716 km north of the study area while September bats had only enough reserves to fly 386 km southward, about 160 km short of the nearest known Sonora wintering locality. It is suggested that in spring the bats may have a more rigidly timed migration and so put on excess fat to counter an uncertain environment to the north. The fall migration may be triggered by more unpredictable events, such as the passage of cold fronts, and less fat reserves may be required for movements into more favorable southern locales.
Pickles, R S A; Groombridge, J J; Zambrana Rojas, V D; Van Damme, P; Gottelli, D; Kundu, S; Bodmer, R; Ariani, C V; Iyengar, A; Jordan, W C
The giant otter, Pteronura brasiliensis, occupies a range including the major drainage basins of South America, yet the degree of structure that exists within and among populations inhabiting these drainages is unknown. We sequenced portions of the mitochondrial DNA (mtDNA) cytochrome b (612bp) and control region (383 bp) genes in order to determine patterns of genetic variation within the species. We found high levels of mtDNA haplotype diversity (h = 0.93 overall) and support for subdivision into four distinct groups of populations, representing important centers of genetic diversity and useful units for prioritizing conservation within the giant otter. We tested these results against the predictions of three hypotheses of Amazonian diversification (Pleistocene Refugia, Paleogeography, and Hydrogeology). While the phylogeographic pattern conformed to the predictions of the Refugia Hypothesis, molecular dating using a relaxed clock revealed the phylogroups diverged from one another between 1.69 and 0.84 Ma, ruling out the influence of Late Pleistocene glacial refugia. However, the role of Plio-Pleistocene climate change could not be rejected. While the molecular dating also makes the influence of geological arches according to the Paleogeography Hypothesis extremely unlikely, the recent Pliocene formation of the Fitzcarrald Arch and its effect of subsequently altering drainage pattern could not be rejected. The data presented here support the interactions of both climatic and hydrological changes resulting from geological activity in the Plio-Pleistocene, in shaping the phylogeographic structure of the giant otter.
Putranto, Riza-Arief; Herlinawati, Eva; Rio, Maryannick; Leclercq, Julie; Piyatrakul, Piyanuch; Gohet, Eric; Sanier, Christine; Oktavia, Fetrina; Pirrello, Julien; Kuswanhadi; Montoro, Pascal
Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD. PMID:26247941
Braicovich, Paola E; Pantoja, Camila; Pereira, Aldenice N; Luque, Jose L; Timi, Juan T
With the aim of evaluating the utility of marine parasites as indicators of zoogeographical regions in the South West Atlantic, we analyzed data on assemblages of long-lived larval parasites of 488 specimens of Percophis brasiliensis distributed in 11 samples from nine localities covering the entire distribution of the species in the Argentine biogeographical Province. Near half a million long-lived parasite individuals belonging to 17 species present in the whole sample displayed clear latitudinal patterns. Data for parasite assemblages at infracommunity and component community levels were analysed in relation to the geographical distance. Significant similarity decay of parasite assemblages over distance was observed, with those based on abundances and mean abundances showing departures from predicted values of regressions. These departures were represented by higher dissimilarities between samples coming from different zoogeographical regions than between those caught within the same region, independently of the distance separating them. Consequently, zoogeographical regions were identified in a distance-decay context. Multivariate analyses corroborated a close fit of similarity between assemblages to existing zoogeographical classifications. Regressions representing distance decay of similarity, and the identification of their outliers, can therefore shed light on the existence of discontinuities or uniformities in the geographic distribution of parasite assemblages and, in turn, in the zoogeography of their fish hosts.
Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric
REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom.
Uthup, Thomas K; Ravindran, Minimol; Bini, K; Thakurdas, Saha
Cytosine methylation is a fundamental epigenetic mechanism for gene-expression regulation and development in plants. Here, we report for the first time the identification of DNA methylation patterns and their putative relationship with abiotic stress in the tree crop Hevea brasiliensis (source of 99% of natural rubber in the world). Regulatory sequences of four major genes involved in the mevalonate pathway (rubber biosynthesis pathway) and one general defense-related gene of three high-yielding popular rubber clones grown at two different agroclimatic conditions were analyzed for the presence of methylation. We found several significant variations in the methylation pattern at core DNA binding motifs within all the five genes. Several consistent clone-specific and location-specific methylation patterns were identified. The differences in methylation pattern observed at certain pivotal cis-regulatory sites indicate the direct impact of stress on the genome and support the hypothesis of site-specific stress-induced DNA methylation. It is assumed that some of the methylation patterns observed may be involved in the stress-responsive mechanism in plants by which they adapt to extreme conditions. The study also provide clues towards the existence of highly divergent phenotypic characters among Hevea clones despite their very similar genetic make-up. Altogether, the observations from this study prove beyond doubt that there exist epigenetic variations in Hevea and environmental factors play a significant role in the induction of site-specific epigenetic mutations in its genome.
Zhang, Yi; Leclercq, Julie; Montoro, Pascal
Environmental stress can lead to oxidative stress resulting from an increase in reactive oxygen species (ROS) and involves redox adjustments. Natural rubber is synthesized in laticifers, which is a non-photosynthetic tissue particularly prone to oxidative stress. This paper reviews the current state of knowledge on the ROS production and ROS-scavenging systems in laticifers. These regulations have been the subject of intense research into a physiological syndrome, called Tapping Panel Dryness (TPD), affecting latex production in Hevea brasiliensis In order to prevent TPD occurrence, monitoring thiol content appeared to be a crucial factor of latex diagnosis. Thiols, ascorbate and γ-tocotrienol are the major antioxidants in latex. They are involved in membrane protection from ROS and likely have an effect on the quality of raw rubber. Some transcription factors might play a role in the redox regulatory network in Hevea, in particular ethylene response factors, which have been the most intensively studied given the role of ethylene on rubber production. Current challenges for rubber research and development with regard to redox systems will involve improving antioxidant capacity using natural genetic variability.
In this study the effect of thermal treatment on the equilibrium moisture content, chemical composition and biological resistance to decay fungi of juvenile and mature Hevea brasiliensis wood (rubber wood) was evaluated. Samples were taken from a 53-year-old rubber wood plantation located in Tabapuã, Sao Paulo, Brazil. The samples were thermally-modified at 180°C, 200°C and 220°C. Results indicate that the thermal modification caused: (1) a significant increase in the extractive content and proportional increase in the lignin content at 220°C; (2) a significant decrease in the equilibrium moisture content, holocelluloses, arabinose, galactose and xylose content, but no change in glucose content; and (3) a significant increase in wood decay resistance against both Pycnoporus sanguineus (L.) Murrill and Gloeophyllum trabeum (Pers.) Murrill decay fungi. The greatest decay resistance was achieved from treatment at 220°C which resulted in a change in wood decay resistance class from moderately resistant to resistant. Finally, this study also demonstrated that the influence of thermal treatment in mature wood was lower than in juvenile wood. PMID:26986200
Gébelin, Virginie; Leclercq, Julie; Kuswanhadi; Argout, Xavier; Chaidamsari, Tetty; Hu, Songnian; Tang, Chaorong; Sarah, Gautier; Yang, Meng; Montoro, Pascal
Natural rubber is harvested by tapping Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. Harvesting stress can lead to tapping panel dryness (TPD). MicroRNAs (miRNAs) are induced by abiotic stress and regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs. This study set out to sequence miRNAs expressed in latex cells and to identify TPD-related putative targets. Deep sequencing of small RNAs was carried out on latex from trees affected by TPD using Solexa technology. The most abundant small RNA class size was 21 nucleotides for TPD trees compared with 24 nucleotides in healthy trees. By combining the LeARN pipeline, data from the Plant MicroRNA database and Hevea EST sequences, we identified 19 additional conserved and four putative species-specific miRNA families not found in previous studies on rubber. The relative transcript abundance of the Hbpre-MIR159b gene increased with TPD. This study revealed a small RNA-specific signature of TPD-affected trees. Both RNA degradation and a shift in miRNA biogenesis are suggested to explain the general decline in small RNAs and, particularly, in miRNAs.
Lercari, D.; Defeo, O.
Sandy beaches are ecosystems which are heavily affected by human activities. An example of this is freshwater discharges, which are known to change salinity, temperature and nutrient regimes and degrade nearshore environments. However, the effects of this kind of disturbance on sandy beach fauna have been little studied. This paper reports the spatial effects of a man-made freshwater canal discharge on the population structure, abundance and reproductive characteristics of the sandy beach mole crab Emerita brasiliensis. Along the 22 km of sandy beach sampled, the mole crab showed a marked longshore variability in population structure and abundance. Abundance of different population components (juveniles, males, females and ovigerous females) significantly decreased towards the canal. Population structure by sex and size, individual weight, fecundity and female maturity patterns at size also displayed a non-linear response to the distance from the freshwater discharge. Only the size structure of males did not follow this pattern. For males, spatial heterogeneity enhanced the detection of density-dependence at less disturbed sites. The authors conclude that artificial freshwater discharges could significantly influence the distribution, abundance and life-history traits of the biota of sandy beaches, and that further study of these ecosystems should include human activities as important factors affecting spatial and temporal trends. The need to consider different spatial and temporal scales in order to detect the effect of anthropogenically-driven impacts in sandy beach populations is stressed.
Nembo, B; Goudey-Perriere, F; Gayral, P; Perriere, C; Brousse-Gaury, P
Specimens of the rat hookworm, Nippostrongylus brasiliensis (Nematoda) were recovered from lungs (third- and fourth-stage larvae) and intestine (fourth-stage larvae and adults). The following features were studied in the different stages by scanning electron microscopy: cephalic structures, especially sense organs, synlophe, cervical region, and caudal part. The main differences between the third and fourth stages concerned the lip-like structures around the oral aperture, the appearance of the cephalic space with the presence of a cephalic cap in fourth-stage larvae, the pattern of longitudinal ridges, and sexual differentiation. Pore-like papillae, not seen in third-stage larvae, developed in later stages. Deirids were observed only in adults, and phasmids were poorly discerned. Some of these morphological features, such as the cephalic sense organ apertures and cuticle pores and micropores, can be observed only by scanning electron microscopy. The possible functions of these different structures and their relationship with the behavior of the worms during their life cycle are discussed.
Putranto, Riza-Arief; Herlinawati, Eva; Rio, Maryannick; Leclercq, Julie; Piyatrakul, Piyanuch; Gohet, Eric; Sanier, Christine; Oktavia, Fetrina; Pirrello, Julien; Kuswanhadi; Montoro, Pascal
Ethephon, an ethylene releaser, is used to stimulate latex production in Hevea brasiliensis. Ethylene induces many functions in latex cells including the production of reactive oxygen species (ROS). The accumulation of ROS is responsible for the coagulation of rubber particles in latex cells, resulting in the partial or complete stoppage of latex flow. This study set out to assess biochemical and histological changes as well as changes in gene expression in latex and phloem tissues from trees grown under various harvesting systems. The Tapping Panel Dryness (TPD) susceptibility of Hevea clones was found to be related to some biochemical parameters, such as low sucrose and high inorganic phosphorus contents. A high tapping frequency and ethephon stimulation induced early TPD occurrence in a high latex metabolism clone and late occurrence in a low latex metabolism clone. TPD-affected trees had smaller number of laticifer vessels compared to healthy trees, suggesting a modification of cambial activity. The differential transcript abundance was observed for twenty-seven candidate genes related to TPD occurrence in latex and phloem tissues for ROS-scavenging, ethylene biosynthesis and signalling genes. The predicted function for some Ethylene Response Factor genes suggested that these candidate genes should play an important role in regulating susceptibility to TPD.
Li, Dejun; Deng, Zhi; Liu, Changren; Zhao, Manman; Guo, Huina; Xia, Zhihui; Liu, Hui
The polyphenol oxidase (PPO) is involved in undesirable browning in many plant foods. Although the PPOs have been studied by several researchers, the isolation and expression profiles of PPO gene were not reported in rubber tree. In this study, a new PPO gene, HbPPO, was isolated from Hevea brasiliensis. The sequence alignment showed that HbPPO indicated high identities to plant PPOs and belonged to dicot branch. The cis-acting regulatory elements related to stress/hormone responses were predicted in the promoter region of HbPPO. Real-time RT-PCR analyses showed that HbPPO expression varied widely depending on different tissues and developmental stages of leaves. Besides being associated with tapping panel dryness, the HbPPO transcripts were regulated by ethrel, wounding, H2O2, and methyl jasmonate treatments. Moreover, the correlation between latex coagulation rate and PPO activity was further confirmed in this study. Our results lay the foundation for further analyzing the function of HbPPO in rubber tree.
Severo, Elias Taylor Durgante; Calonego, Fred Willians; Sansígolo, Cláudio Angeli; Bond, Brian
In this study the effect of thermal treatment on the equilibrium moisture content, chemical composition and biological resistance to decay fungi of juvenile and mature Hevea brasiliensis wood (rubber wood) was evaluated. Samples were taken from a 53-year-old rubber wood plantation located in Tabapuã, Sao Paulo, Brazil. The samples were thermally-modified at 180°C, 200°C and 220°C. Results indicate that the thermal modification caused: (1) a significant increase in the extractive content and proportional increase in the lignin content at 220°C; (2) a significant decrease in the equilibrium moisture content, holocelluloses, arabinose, galactose and xylose content, but no change in glucose content; and (3) a significant increase in wood decay resistance against both Pycnoporus sanguineus (L.) Murrill and Gloeophyllum trabeum (Pers.) Murrill decay fungi. The greatest decay resistance was achieved from treatment at 220°C which resulted in a change in wood decay resistance class from moderately resistant to resistant. Finally, this study also demonstrated that the influence of thermal treatment in mature wood was lower than in juvenile wood.
Almeida-Paes, Rodrigo; Figueiredo-Carvalho, Maria Helena Galdino; Brito-Santos, Fábio; Almeida-Silva, Fernando; Oliveira, Manoel Marques Evangelista; Zancopé-Oliveira, Rosely Maria
Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies.
Hurley, L M; Thompson, A M
Anatomical and electrophysiological evidence suggests that serotonin alters the processing of sound in the auditory brainstem of many mammalian species. The Mexican free-tailed bat is a hearing specialist, like other microchiropteran bats. At the same time, many aspects of its auditory brainstem are similar to those in other mammals. This dichotomy raises an interesting question regarding the serotonergic innervation of the bat auditory brainstem: Is the serotonergic input to the auditory brainstem similar in bats and other mammals, or are there specializations in the serotonergic innervation of bats that may be related to their exceptional hearing capabilities? To address this question, we immunocytochemically labeled serotonergic fibers in the brainstem of the Mexican free-tailed bat, Tadarida brasiliensis. We found many similarities in the pattern of serotonergic innervation of the auditory brainstem in Tadarida compared with other mammals, but we also found two striking differences. Similarities to staining patterns in other mammals included a higher density of serotonergic fibers in the dorsal cochlear nucleus and in granule cell regions than in the ventral cochlear nucleus, a high density of fibers in some periolivary nuclei of the superior olive, and a higher density of fibers in peripheral regions of the inferior colliculus compared with its core. The two novel features of serotonergic innervation in Tadarida were a high density of fibers in the fusiform layer of the dorsal cochlear nucleus relative to surrounding layers and a relatively high density of serotonergic fibers in the low-frequency regions of the lateral and medial superior olive.
Almeida-Paes, Rodrigo; Figueiredo-Carvalho, Maria Helena Galdino; Brito-Santos, Fábio; Almeida-Silva, Fernando; Oliveira, Manoel Marques Evangelista; Zancopé-Oliveira, Rosely Maria
Terbinafine is a recommended therapeutic alternative for patients with sporotrichosis who cannot use itraconazole due to drug interactions or side effects. Melanins are involved in resistance to antifungal drugs and Sporothrix species produce three different types of melanin. Therefore, in this study we evaluated whether Sporothrix melanins impact the efficacy of antifungal drugs. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) of two Sporothrix brasiliensis and four Sporothrix schenckii strains grown in the presence of the melanin precursors L-DOPA and L-tyrosine were similar to the MIC determined by the CLSI standard protocol for S. schenckii susceptibility to amphotericin B, ketoconazole, itraconazole or terbinafine. When MICs were determined in the presence of inhibitors to three pathways of melanin synthesis, we observed, in four strains, an increase in terbinafine susceptibility in the presence of tricyclazole, a DHN-melanin inhibitor. In addition, one S. schenckii strain grown in the presence of L-DOPA had a higher MFC value when compared to the control. Growth curves in presence of 2×MIC concentrations of terbinafine showed that pyomelanin and, to a lesser extent, eumelanin were able to protect the fungi against the fungicidal effect of this antifungal drug. Our results suggest that melanin protects the major pathogenic species of the Sporothrix complex from the effects of terbinafine and that the development of new antifungal drugs targeting melanin synthesis may improve sporotrichosis therapies. PMID:27031728
Omar, Atef; Foissner, Wilhelm
Using standard morphological methods, we describe one new Leptopharynx species and a new subspecies of L. costatus, both from soil of the neotropic region. Further, we studied two populations of L. costatus costatus. Leptopharynx brasiliensis nov. spec., which was discovered in the Mato Grosso, Brazil, is a large member (60μm) of the genus with an enormous oral basket. It differs from similar congeners in having six monokinetids in kinety 6, widely spaced kinetids in kinety 1, and an average of 294 kinetids. Leptopharynx costatus gonohymen nov. subspec., which was discovered in southern Florida, makes a small (35μm) and a large morph (55μm) both with narrow oral basket. The small morph is inseparable from the small morph of L. costatus costatus, while the large morph has right-angled adoral membranelles and widely (vs. narrowly) spaced kinetids in kinety 1. The small morphs of a Brazilian and an Austrian L. costatus match Mexican and other European populations, all having on average 181-187 kinetids. As yet, we know four morphs of L. costatus that differ by body size (small vs. large), the oral basket (narrow vs. wide), membranelle 1 (present vs. absent), and the arrangement of the membranelles (flat vs. angled).
Stephens, R J; Cabral-Anderson, L J
The process of erythropoiesis and vasculogenesis in the yolk sac of the bat (Tadarida brasiliensis cynocephala) has been studied through the use of both light and electron microscopy. Stem cells arise from the leading edge of the migrating splanchnic mesoderm and transform into primitive erythroblasts. Differentiation involves either contact or association with the endodermal cells, since all erythropoietic activity occurs on the endodermal side of the expanding vascular bed, and many of the cells are in close apposition to the lateral or basal plasma membranes of the endodermal cells. Endodermal cells also phagocytize developing primitive erythroblasts during the later stage of the process when erythropoiesis is subsiding in the yolk sac. Cells destined to become the endothelium of the expanding vascular bed also arise from the leading edge of the migrating splanchnic mesoderm. Their process of differentiation involves the development of cytoplasmic extensions that may surround a group of differentiating erythroblasts, enclosing them in the newly formed lumen of the blood vessel. The cytoplasmic extensions make contact and develop junctional complexes with similar processes from other cells to complete the lumen of the lengthening vascular bed. Cells of the granulocyte series or megakaryocytes are not observed in the yolk sac of the bat as has been described in certain other species.
Clark, D R
DDT is believed to have caused the population of Brazilian free-tailed bats (Tadarida brasiliensis mexicana) at Carlsbad Cavern to decline severely after 1936. Nevertheless, previous data supporting this hypothesis are limited to a single study from 1974, which indicated that 20% of young free-tails from the cavern may have died of DDE poisoning during their first southward migration. In this study I compared organochlorine residues among samples of free-tails collected in Carlsbad Cavern in 1930, 1956, 1965, 1973, and 1988. Samples of skin cut from dry museum specimens were chemically analyzed, except for the 1973 data, which were derived from analyses of whole bats minus gastrointestinal tracts. Accumulated residue levels of DDT compounds in bats from 1965 and 1956 exceeded those in 1973 bats by approximately 4.8 times and approximately 2.7 times, respectively. This suggests that lethal effects of DDT compounds were substantially greater in the 1950s and 1960s than in the 1970s. Residues in 1988 bats resembled those for 1973 bats. It is concluded that DDT played a major role in this severe population decline. These results can be applied by management personnel in evaluating the present and future status of this population regarding persisting organochlorine insecticides as well as other agricultural chemicals now in use. The case of the Carlsbad colony is discussed relative to the general issue of other bat population declines.
Russell, A L; Medellín, R A; McCracken, G F
Incomplete lineage sorting can genetically link populations long after they have diverged, and will exert a more powerful influence on larger populations. The effects of this stochastic process can easily be confounded with those of gene flow, potentially leading to inaccurate estimates of dispersal capabilities or erroneous designation of evolutionarily significant units (ESUs). We have used phylogenetic, population genetic, and coalescent methods to examine genetic structuring in large populations of a widely dispersing bat species and to test hypotheses concerning the influences of coalescent stochasticity vs. gene flow. The Mexican free-tailed bat, Tadarida brasiliensis mexicana, exhibits variation in both migratory tendency and route over its range. Observations of the species' migratory behaviour have led to the description of behaviourally and geographically defined migratory groups, with the prediction that these groups compose structured gene pools. Here, we used mtDNA sequence analyses coupled with existing information from allozyme, banding, and natural history studies to evaluate hypotheses regarding the relationship between migration and genetic structure. Analyses of molecular variance revealed no significant genetic structuring of behaviourally distinct migratory groups. Demographic analyses were consistent with population growth, although the timing of population expansion events differs between migratory and nonmigratory populations. Hypotheses concerning the role of gene flow vs. incomplete lineage sorting on these data are explored using coalescent simulations. Our study demonstrates the importance of accounting for coalescent stochasticity in formulating phylogeographical hypotheses, and indicates that analyses that do not take such processes into account can lead to false conclusions regarding a species' phylogeographical history.
Clark, D R; Kroll, J C
Adult female free-tailed bats (Tadarida brasiliensis) were collected at Bracken Cave, Texas, and shipped to the Patuxent Wildlife Research Center. Treated mealworms (Tenebrio molitor) containing 107 ppm DDE were fed to 17 bats; five other bats were fed untreated mealworms. After 40 days on dosage, during which one dosed bat was killed accidentally, four dosed bats were frozen and the remaining 17 were starved to death. The objective was to elevate brain levels of DDE to lethality and measure these concentrations. After the feeding period, dosed bats weighed less than controls. After starvation, the body condition of dosed bats was poorer than that of controls even though there was no difference in the amounts of carcass fat. During starvation, dosed bats lost weight faster than controls. Also, four dosed bats exhibited the prolonged tremoring that characterizes DDE poisoning. DDE increased in brains of starving bats as fat was metabolized. The estimated mean brain concentration of DDE diagnostic of death was 519 ppm with a range of 458-564 ppm. These values resemble diagnostic levels known for two species of passerine birds, but they exceed published levels for two free-tailed bats from Carlsbad Caverns, New Mexico.
Clark, D.R.; Kroll, J.C.
Adult female free-tailed bats (Tadarida brasiliensis) were collected at Bracken Cave, Texas, and shipped to the Patuxent Wildlife Research Center. Treated mealworms (Tenebrio molitor) containing 107 ppm DDE were fed to 17 bats; five other bats were fed untreated mealworms. After 40 days on dosage, during which one dosed bat was killed accidentally, four dosed bats were frozen and the remaining 17 were starved to death. The objective was to elevate brain levels of DDE to lethality and measure these concentrations. After the feeding period, dosed bats weighed less than controls. After starvation, the body condition of dosed bats was poorer than that of controls even though there was no difference in the amounts of carcass fat. During starvation, dosed bats lost weight faster than controls. Also, four dosed bats exhibited the prolonged tremoring that characterizes DDE poisoning. DDE increased in brains of starving bats as fat was metabolized. The estimated mean brain concentration of DDE diagnostic of death was 519 ppm with a range of 458-564 ppm. These values resemble diagnostic levels known for two species of passerine birds, but they exceed published levels for two free-tailed bats from Carlsbad Caverns, New Mexico.
Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Coulary-Salin, Bénédicte; Bentaleb, Ahmed; Cullin, Christophe; Deffieux, Alain; Peruch, Frédéric
REF (Hevb1) and SRPP (Hevb3) are two major components of Hevea brasiliensis latex, well known for their allergenic properties. They are obviously taking part in the biosynthesis of natural rubber, but their exact function is still unclear. They could be involved in defense/stress mechanisms after tapping or directly acting on the isoprenoid biosynthetic pathway. The structure of these two proteins is still not described. In this work, it was discovered that REF has amyloid properties, contrary to SRPP. We investigated their structure by CD, TEM, ATR-FTIR and WAXS and neatly showed the presence of β-sheet organized aggregates for REF, whereas SRPP mainly fold as a helical protein. Both proteins are highly hydrophobic but differ in their interaction with lipid monolayers used to mimic the monomembrane surrounding the rubber particles. Ellipsometry experiments showed that REF seems to penetrate deeply into the monolayer and SRPP only binds to the lipid surface. These results could therefore clarify the role of these two paralogous proteins in latex production, either in the coagulation of natural rubber or in stress-related responses. To our knowledge, this is the first report of an amyloid formed from a plant protein. This suggests also the presence of functional amyloid in the plant kingdom. PMID:23133547
Mendonça, Mônica Sawan; Peraçolli, Terezinha S; Silva-Vergara, Mário León; Ribeiro, Sílvio C; Oliveira, Rafael Faria; Mendes, Rinaldo Poncio; Rodrigues, Virmondes
Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp. (p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4 promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp. infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4 gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described. PMID:26517657
Agbo, Matthias Onyebuchi; Nworu, Chukwuemeka Sylvester; Okoye, Festus Basden Chied; Osadebe, Patience Ogoamaka
The present study was carried out to evaluate the anti-inflammatory activities of polyphenols isolated from the leaves of mistletoe (Loranthus micranthus Linn.) parasitic on Hevea brasiliensis. The anti-inflammatory properties of the isolated compounds were evaluated on the basis of their ability to inhibit the production of nitric oxide (NO) and tumuor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS) activated RAW 264.7 mouse macrophages. Semi-preparative HPLC separation of the ethyl acetate (EtOAc) and butanol (n-BuOH) fractions of the leaves of mistletoe (Loranthus micranthus Linn) parasitic on Hevea brasiliensis led to the isolation of four polyphenols: 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG) (1); (-)-epicatechin-3-O-(3″-O-methyl)-gallate (ECG3″Me) (2); rutin (3) and peltatoside (4). Compounds 1-4 were isolated for the first time from this plant while 1 was isolated for the first time in nature. These compounds (1-4) were readily identified by comparison of their spectroscopic data with those reported in the literature. The polyphenols proved to have anti-inflammatory activity as evidenced by the suppression of inducible nitric oxide (iNO) and cytokine (TNF-α) levels in the culture supernatant of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. However, the study showed that the quercetin diglycosides showed stronger inhibition of proinflammatory mediators than the epicatechin derivates. These data provide evidence that polyphenolic compounds isolated from the mistletoe parasitic on Hevea brasiliensis may contribute to its anti-inflammatory properties by inhibiting the expression of inducible nitric oxide and proinflammatory cytokines such as tumour necrosis factor-α. PMID:26417309
Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira
Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.
Fang, Leilei; Zhang, Yanqing; Xie, Junbo; Wang, Lijuan; Zhang, Huan; Wei, Weilu; Li, Yingrui
The royal sun mushroom, Agaricus brasiliensis is a widely consumed mushroom around the world. In this study, the immunoregulatory potential of A. brasiliensis polysaccharides was investigated in vitro and in vivo. In vivo, the polysaccharides remarkably increased the spleen and thymus indexes in mice, and this effect was influenced significantly by age (the adult and the juvenile). The spleen index increased by 27.28% in adult mice treated with the polysaccharides, whereas the increase in juvenile mice was just 12.59% at the dose of 150 mg·kg-1·d-1. Moreover, the effect of the polysaccharides on the thymus and spleen indexes in adult mice was obvious both in males and females. The carbon clearance ability (phagocytic index) was improved with increasing doses, (32.81% at 120 mg·kg-1·d-1, and 38.34% at 150 mg·kg-1·d-1) in mice treated with the polysaccharides. In vitro, the polysaccharides increased the RAW264.7 cell proliferation with 34.78% at 25 µg/mL and 26.78% at 50 µg/mL. Furthermore, the polysaccharides also promoted mRNA expressions of interleukin (IL)-6, IL-1β, cyclooxygenase-2, and Toll-like receptor 4 (TLR4), myeloid differentiation 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF) in the cells, indicating that the polysaccharides induce the secretion of inflammatory cytokines by stimulating TLR4/MyD88 and TLR4/TRIF pathways. In conclusion, these results suggest that A. brasiliensis polysaccharides induce a very promising immunostimulation effect in vivo and in vitro. Therefore, it should be explored as a novel natural functional food additive.
Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira
Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection. PMID:25048025
Taylor, Maria Lucia; Chávez-Tapia, Catalina B; Rojas-Martínez, Alberto; del Rocio Reyes-Montes, Maria; del Valle, Mirian Bobadilla; Zúñiga, Gerardo
Fourteen Histoplasma capsulatum isolates recovered from infected bats captured in Mexican caves and two human H. capsulatum reference strains were analyzed using random amplification of polymorphic DNA PCR-based and partial DNA sequences of four genes. Cluster analysis of random amplification of polymorphic DNA-patterns revealed differences for two H. capsulatum isolates of one migratory bat Tadarida brasiliensis. Three groups were identified by distance and maximum-parsimony analyses of arf, H-anti, ole, and tub1 H. capsulatum genes. Group I included most isolates from infected bats and one clinical strain from central Mexico; group II included the two isolates from T. brasiliensis; the human G-217B reference strain from USA formed an independent group III. Isolates from group II showed diversity in relation to groups I and III, suggesting a different H. capsulatum population.
Lee, D L
The ultrastructure of the excretory system, including the subventral glands, of the nematode Nippostrongylus brasiliensis has been described. The walls of the lateral excretory canals contain canaliculi which open into the lumen of the canal. It is suggested that these canals play a role in osmoregulation and excretion. The sub-ventral glands contain two types of secretory granule and contain non-specific esterase, cholinesterase and aminopeptidase. It is suggested that these glands are not excretory but play an important role in feeding.
Giambelluca, Thomas W.; Mudd, Ryan G.; Liu, Wen; Ziegler, Alan D.; Kobayashi, Nakako; Kumagai, Tomo'omi; Miyazawa, Yoshiyuki; Lim, Tiva Khan; Huang, Maoyi; Fox, Jefferson; Yin, Song; Mak, Sophea Veasna; Kasemsap, Poonpipope
To investigate the effects of expanding rubber (Hevea brasiliensis) cultivation on water cycling in Mainland Southeast Asia (MSEA), evapotranspiration (ET) was measured within rubber plantations at Bueng Kan, Thailand, and Kampong Cham, Cambodia. After energy closure adjustment, mean annual rubber ET was 1211 and 1459 mm yr-1 at the Thailand and Cambodia sites, respectively, higher than that of other tree-dominated land covers in the region, including tropical seasonal forest (812-1140 mm yr-1), and savanna (538-1060 mm yr-1). The mean proportion of net radiation used for ET by rubber (0.725) is similar to that of tropical rainforest (0.729) and much higher than that of tropical seasonal forest (0.595) and savanna (0.548). Plant area index (varies with leaf area changes), explains 88.2% and 73.1% of the variance in the ratio of latent energy flux (energy equivalent of ET) to potential latent energy flux (LE/LEpot) for midday rain-free periods at the Thailand and Cambodia sites, respectively. High annual rubber ET results from high late dry season water use, associated with rapid refoliation by this brevideciduous species, facilitated by tapping of deep soil water, and by very high wet season ET, a characteristic of deciduous trees. Spatially, mean annual rubber ET increases strongly with increasing net radiation (Rn) across the three available rubber plantation observation sites, unlike nonrubber tropical ecosystems, which reduce canopy conductance at high Rn sites. High water use by rubber raises concerns about potential effects of continued expansion of tree plantations on water and food security in MSEA.
Clark, D.R.; Martin, C.O.; Swineford, D.M.
Fifty-nine free-tailed bats (Tadarida brasiliensis mexicana ) were collected at Bracken Cave, Texas, and analyzed for organochlorine insecticides and polychlorinated biphenyls (PCBs). Residues of DDE in the brain were greater in 12 young collected from the floor than in 15 young taken from the ceiling, but food deprivation, not higher residues in the brain, apparently caused young to fall....Among 18 pregnant females, residues of DDE and DDT were highest in yearlings. The first lactation by yearlings caused their residue loads to drop sharply. Thereafter, increasing age was accompanied by increasing residues but amounts generally did not exceed those in yearlings.....Residue levels in embryos were a function both of levels in the female parent and degree of embryonic development. Residues accumulated rapidly in nursing young, and lactating females may excrete from 1.3 to 16.2 (mean = 4.3) micrograms of DDE in milk per day. Maximum individual residue loads may be attained toward the end of nursing, and mobilization of these residues during southward migration may subject Bracken Cave free-tails to maximum lifetime residues in the brain....Comparison of our data with residue data for the free-tail population at Eagle Creek Cave (Arizona) in 1970 produced the following conclusions: ( 1) residues of DDE appeared similar in pregnant females, embryos, lactating females, and fallen young for the two populations; (2) residues of DDT and dieldrin appeared greater in pregnant females at Bracken Cave; (3) DDE and DDT occurred at greater levels in guano samples from Bracken Cave. On this basis, the population decline observed at Eagle Creek Cave between 1963 and 1969 does not appear to be related to the residues observed in the 1970 samples taken from that cave.
Beloti, Lilian L; Costa, Bruna Z; Toledo, Marcelo A S; Santos, Clelton A; Crucello, Aline; Fávaro, Marianna T P; Santiago, André S; Mendes, Juliano S; Marsaioli, Anita J; Souza, Anete P
A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.
Chaudhuri, P S; Bhattacharjee, Subhalaxmi; Dey, Animesh; Chattopadhyay, Sharmila; Bhattacharya, Dipto
A comparative analysis of earthworm communities was carried out in the rubber plantations (Hevea brasiliensis) of different age groups in West Tripura to understand the impact of such exotic and monoculture plantation in biodiversity conservation. Earthworm communities were studied on monthly basis over a period of one year (2006-2007) in the 3, 10, 14, 20 and 25 year-old plantations. Among twelve earthworm species collected from the studied sites, six species belonged to Octochaetidae [Eutyphoeus assomensis Stephenson, Eutyphoeus comillahnus Michaelsen, Lennogaster chittagongensis (Stephensen), Octochaetona beatrix Gates, Dichogaster offinis Michaelsen, Lennogaster yeicus (Stephensen)], two species each to Megascolecidae [Metaphire houlleti (Perrier), Konchurio sp. 1] and Moniligastridae [Drowida nepalensis Michaelsen, Drawida papillifer papillifer Stephenson], one species each to Glossoscolecidae [Pontoscolex corethrurus (Muller)] and Ocnerodrilidae [Gordiodrilus elegans Beddard]. Exotic species P corethrurus, M. houlleti and native peregrine species like D. nepolensis and D. papillifer papillifer were distributed in all the age groups of plantation, while other species showed restricted distribution. P. corethrurus contributed more than 60% biomass and 70% density of earthworm communities in rubber plantation. With aging of rubber plantations both the densities and biomasses of earthworms increased. High contents of polyphenol, flavonoid and lignin in the litters of 3 and 10 year-old-rubber plantations through their effects on food intake, probably resulted to low biomass values of earthworms in those age groups of plantation. With further increase in the age of plantations beyond 10 years, polyphenol, flavonoid and lignin contents decreased. Accordingly the biomass of earthworms increased with increase in the age of plantation. Soil moisture increased with increase in the age of plantation and there was a good positive correlation between soil moisture and
Light is one of most important factors to plants because it is necessary for photosynthesis. In this study, physiological and gene expression analyses under different light intensities were performed in the seedlings of rubber tree (Hevea brasiliensis) clone GT1. When light intensity increased from 20 to 1000 µmol m(-2) s(-1), there was no effect on the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), indicating that high light intensity did not damage the structure and function of PSII reaction center. However, the effective photochemical quantum yield of PSII (Y(II)), photochemical quenching coefficient (qP), electron transfer rate (ETR), and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (qL) were increased significantly as the light intensity increased, reached a maximum at 200 µmol m(-2) s(-1), but decreased from 400 µmol m(-2) s(-1). These results suggested that the PSII photochemistry showed an optimum performance at 200 µmol m(-2) s(-1) light intensity. The chlorophyll content was increased along with the increase of light intensity when it was no more than 400 µmol m(-2) s(-1). Since increasing light intensity caused significant increase in H2O2 content and decreases in the per unit activity of antioxidant enzymes SOD and POD, but the malondialdehyde (MDA) content was preserved at a low level even under high light intensity of 1000 µmol m(-2) s(-1), suggesting that high light irradiation did not induce membrane lipid peroxidation in rubber tree. Moreover, expressions of antioxidant-related genes were significantly up-regulated with the increase of light intensity. They reached the maximum expression at 400 µmol m(-2) s(-1), but decreased at 1000 µmol m(-2) s(-1). In conclusion, rubber tree could endure strong light irradiation via a specific mechanism. Adaptation to high light intensity is a complex process by regulating antioxidant enzymes activities, chloroplast formation, and
Cardozo, F. T. G. S.; Camelini, C. M.; Cordeiro, M. N. S.; Mascarello, A.; Malagoli, B. G.; Larsen, I.; Rossi, M. J.; Nunes, R. J.; Braga, F. C.; Brandt, C.R.; Simões, C. M. O.
Agaricus brasiliensis cell-wall polysaccharides isolated from fruiting body (FR) and mycelium (MI) and their respective sulfated derivatives (FR-S and MI-S) were chemically characterized using elemental analysis, TLC, FT-IR, NMR, HPLC, and thermal analysis. Cytotoxic activity was evaluated against A549 tumor cells by MTT and sulforhodamine assays. The average molecular weight (Mw) of FR and MI was estimated to be 609 and 310 kDa, respectively. FR-S (127 kDa) and MI-S (86 kDa) had lower Mw, probably due to hydrolysis occurred during the sulfation reaction. FR-S and MI-S presented ~14 % sulfur content in elemental analysis. Sulfation of samples was characterized by the appearance of two new absorption bands at 1253 and 810 cm−1 in the infrared spectra, related to S=O and C-S-O sulfate groups, respectively. Through 1H and 13C NMR analysis FR-S was characterized as a (1→6)-(1→3)-β-D-glucan fully sulfated at C-4 and C-6 terminal and partially sulfated at C-6 of (1→3)-β-D-glucan moiety. MI-S was shown to be a (1→3)-β-D-gluco-(1→2)-β-D-mannan, partially sulfated at C-2, C-3, C-4, and C-6, and fully sulfated at C-6 of the terminal residues. The combination of high degree of sulfation and low molecular weight was correlated with the increased cytotoxic activity (48 h of treatment) of both FR-S (EC50=605.6 μg/mL) and MI-S (EC50=342.1 μg/mL) compared to the non-sulfated polysaccharides FR and MI (EC50>1500 μg/mL). PMID:23511057
Background Plants respond to external stimuli through fine regulation of gene expression partially ensured by small RNAs. Of these, microRNAs (miRNAs) play a crucial role. They negatively regulate gene expression by targeting the cleavage or translational inhibition of target messenger RNAs (mRNAs). In Hevea brasiliensis, environmental and harvesting stresses are known to affect natural rubber production. This study set out to identify abiotic stress-related miRNAs in Hevea using next-generation sequencing and bioinformatic analysis. Results Deep sequencing of small RNAs was carried out on plantlets subjected to severe abiotic stress using the Solexa technique. By combining the LeARN pipeline, data from the Plant microRNA database (PMRD) and Hevea EST sequences, we identified 48 conserved miRNA families already characterized in other plant species, and 10 putatively novel miRNA families. The results showed the most abundant size for miRNAs to be 24 nucleotides, except for seven families. Several MIR genes produced both 20-22 nucleotides and 23-27 nucleotides. The two miRNA class sizes were detected for both conserved and putative novel miRNA families, suggesting their functional duality. The EST databases were scanned with conserved and novel miRNA sequences. MiRNA targets were computationally predicted and analysed. The predicted targets involved in "responses to stimuli" and to "antioxidant" and "transcription activities" are presented. Conclusions Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and novel miRNAs when the complete genome is not yet available. Our study provided additional information for evolutionary studies and revealed potentially specific regulation of the control of redox status in Hevea. PMID:22330773
Background Rubber tree (Hevea brasiliensis) laticifers are the source of natural rubber. Rubber production depends on endogenous and exogenous ethylene (ethephon). AP2/ERF transcription factors, and especially Ethylene-Response Factors, play a crucial role in plant development and response to biotic and abiotic stresses. This study set out to sequence transcript expressed in various tissues using next-generation sequencing and to identify AP2/ERF superfamily in the rubber tree. Results The 454 sequencing technique was used to produce five tissue-type transcript libraries (leaf, bark, latex, embryogenic tissues and root). Reads from all libraries were pooled and reassembled to improve mRNA lengths and produce a global library. One hundred and seventy-three AP2/ERF contigs were identified by in silico analysis based on the amino acid sequence of the conserved AP2 domain from the global library. The 142 contigs with the full AP2 domain were classified into three main families (20 AP2 members, 115 ERF members divided into 11 groups, and 4 RAV members) and 3 soloist members. Fifty-nine AP2/ERF transcripts were found in latex. Alongside the microRNA172 already described in plants, eleven additional microRNAs were predicted to inhibit Hevea AP2/ERF transcripts. Conclusions Hevea has a similar number of AP2/ERF genes to that of other dicot species. We adapted the alignment and classification methods to data from next-generation sequencing techniques to provide reliable information. We observed several specific features for the ERF family. Three HbSoloist members form a group in Hevea. Several AP2/ERF genes highly expressed in latex suggest they have a specific function in Hevea. The analysis of AP2/ERF transcripts in Hevea presented here provides the basis for studying the molecular regulation of latex production in response to abiotic stresses and latex cell differentiation. PMID:23324139
Richter, Sven; Nieveler, Jens; Schulze, Holger; Bachmann, Till T; Schmid, Rolf D
The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.
Cardozo, F T G S; Camelini, C M; Cordeiro, M N S; Mascarello, A; Malagoli, B G; Larsen, I V; Rossi, M J; Nunes, R J; Braga, F C; Brandt, C R; Simões, C M O
Agaricus brasiliensis cell-wall polysaccharides isolated from fruiting body (FR) and mycelium (MI) and their respective sulfated derivatives (FR-S and MI-S) were chemically characterized using elemental analysis, TLC, FT-IR, NMR, HPLC, and thermal analysis. Cytotoxic activity was evaluated against A549 tumor cells by MTT and sulforhodamine assays. The average molecular weight (Mw) of FR and MI was estimated to be 609 and 310 kDa, respectively. FR-S (127 kDa) and MI-S (86 kDa) had lower Mw, probably due to hydrolysis occurring during the sulfation reaction. FR-S and MI-S presented ~14% sulfur content in elemental analysis. Sulfation of samples was characterized by the appearance of two new absorption bands at 1253 and 810 cm(-1) in the infrared spectra, related to S=O and C-S-O sulfate groups, respectively. Through (1)H and (13)C NMR analysis FR-S was characterized as a (1→6)-(1→3)-β-D-glucan fully sulfated at C-4 and C-6 terminal and partially sulfated at C-6 of (1→3)-β-D-glucan moiety. MI-S was shown to be a (1→3)-β-D-gluco-(1→2)-β-D-mannan, partially sulfated at C-2, C-3, C-4, and C-6, and fully sulfated at C-6 of the terminal residues. The combination of high degree of sulfation and low molecular weight was correlated with the increased cytotoxic activity (48 h of treatment) of both FR-S (EC₅₀=605.6 μg/mL) and MI-S (EC₅₀=342.1 μg/mL) compared to the non-sulfated polysaccharides FR and MI (EC₅₀>1500 μg/mL).
Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet
Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.
Cheng, Chun-Man; Wang, Ru-Song; Jiang, Ju-Sheng
The development of rubber industry depends on the sustainable management of rubber plantation. To evaluate the environmental effects of planting Hevea brasiliensis on a subsystem of tropical forest ecosystem, the variation of soil fertility and carbon sequestration under rubber plantation within 30-year life period were investigated in Hainan Island. Results showed that (1) with the increase of stand age of rubber plantation, soil fertility decreased all along. From 1954 to 1995, soil organic matter, total N, available K and available P decreased by 48.2%, 54.1%, 56.7% and 64.1%, respectively. (2) If the complete return of litters was considered without additional fertilizer application to the soil of the rubber plantations, the consumption periods for P, N, K, Mg were only 825 years, 329 years, 94 years and 65 years, respectively. To improve soil fertility is essential for rubber plantation development. (3) The C sequestration of rubber trees per hectare accounts for 272.08 t within 30-year life period and 57.91% of them was fixed in litters. In comparison with C sequestration by rain forest (234.305 t/hm2) and by secondary rain forest (150.203 t/hm2), rubber forest has more potentials for C fixation. On the base of above results, the following measures would benefit the maintenance of soil fertility and the development of rubber industry, including applying fertilizer to maintain the balance of soil nutrients, intercropping leguminous plant to improve soil fertility, reducing the collection of litters, optimizing soil properties to improve element P availability such as applying CaCO3. The information gathered from the study can be used as baseline data for the sustainable management of rubber plantation elsewhere.
Putranto, Riza-Arief; Duan, Cuifang; Kuswanhadi; Chaidamsari, Tetty; Rio, Maryannick; Piyatrakul, Piyanuch; Herlinawati, Eva; Pirrello, Julien; Dessailly, Florence; Leclercq, Julie; Bonnot, François; Tang, Chaorong; Hu, Songnian; Montoro, Pascal
Tolerance of recurrent mechanical wounding and exogenous ethylene is a feature of the rubber tree. Latex harvesting involves tapping of the tree bark and ethephon is applied to increase latex flow. Ethylene is an essential element in controlling latex production. The ethylene signalling pathway leads to the activation of Ethylene Response Factor (ERF) transcription factors. This family has been identified in Hevea brasiliensis. This study set out to understand the regulation of ERF genes during latex harvesting in relation to abiotic stress and hormonal treatments. Analyses of the relative transcript abundance were carried out for 35 HbERF genes in latex, in bark from mature trees and in leaves from juvenile plants under multiple abiotic stresses. Twenty-one HbERF genes were regulated by harvesting stress in laticifers, revealing an overrepresentation of genes in group IX. Transcripts of three HbERF-IX genes from HbERF-IXc4, HbERF-IXc5 and HbERF-IXc6 were dramatically accumulated by combining wounding, methyl jasmonate and ethylene treatments. When an ethylene inhibitor was used, the transcript accumulation for these three genes was halted, showing ethylene-dependent induction. Subcellular localization and transactivation experiments confirmed that several members of HbERF-IX are activator-type transcription factors. This study suggested that latex harvesting induces mechanisms developed for the response to abiotic stress. These mechanisms probably depend on various hormonal signalling pathways. Several members of HbERF-IX could be essential integrators of complex hormonal signalling pathways in Hevea.
Liu, Hui; Deng, Zhi; Chen, Jiangshu; Wang, Sen; Hao, Lili; Li, Dejun
Metacaspases, a family of cysteine proteases, have been suggested to play important roles in programmed cell death (PCD) during plant development and stress responses. To date, no systematic characterization of this gene family has been reported in rubber tree (Hevea brasiliensis). In the present study, nine metacaspase genes, designated as HbMC1 to HbMC9, were identified from whole-genome sequence of rubber tree. Multiple sequence alignment and phylogenetic analyses suggested that these genes were divided into two types: type I (HbMC1-HBMC7) and type II (HbMC8 and HbMC9). Gene structure analysis demonstrated that type I and type II HbMCs separately contained four and two introns, indicating the conserved exon-intron organization of HbMCs. Quantitative real-time PCR analysis revealed that HbMCs showed distinct expression patterns in different tissues, suggesting the functional diversity of HbMCs in various tissues during development. Most of the HbMCs were regulated by drought, cold, and salt stress, implying their possible functions in regulating abiotic stress-induced cell death. Of the nine HbMCs, HbMC1, HbMC2, HbMC5, and HbMC8 displayed a significantly higher relative transcript accumulation in barks of tapping panel dryness (TPD) trees compared with healthy trees. In addition, the four genes were up-regulated by ethephon (ET) and methyl jasmonate (MeJA), indicating their potential involvement in TPD resulting from ET- or JA-induced PCD. In summary, this work provides valuable information for further functional characterization of HbMC genes in rubber tree.
Uthup, T K; Rajamani, A; Ravindran, M; Saha, T
Hydroxy-methylglutaryl coenzyme-A synthase (HMGS) is a rate-limiting enzyme in the cytoplasmic isoprenoid biosynthesis pathway leading to natural rubber production in Hevea brasiliensis (rubber). Analysis of the structural variants of this gene is imperative to understand their functional significance in rubber biosynthesis so that they can be properly utilised for ongoing crop improvement programmes in Hevea. We report here allele richness and diversity of the HMGS gene in selected popular rubber clones. Haplotypes consisting of single nucleotide polymorphisms (SNPs) from the coding and non-coding regions with a high degree of heterozygosity were identified. Segregation and linkage disequilibrium analysis confirmed that recombination is the major contributor to the generation of allelic diversity, rather than point mutations. The evolutionarily conserved nature of some SNPs was identified by comparative DNA sequence analysis of HMGS orthologues from diverse taxa, demonstrating the molecular evolution of rubber biosynthesis genes in general. In silico three-dimensional structural studies highlighting the structural positioning of non-synonymous SNPs from different HMGS haplotypes revealed that the ligand-binding site on the enzyme remains impervious to the reported sequence variations. In contrast, gene expression results indicated the possibility of association between specific haplotypes and HMGS expression in Hevea clones, which may have a downstream impact up to the level of rubber production. Moreover, haplotype diversity of the HMGS gene and its putative association with gene expression can be the basis for further genetic association studies in rubber. Furthermore, the data also show the role of SNPs in the evolution of candidate genes coding for functional traits in plants.
Light is one of most important factors to plants because it is necessary for photosynthesis. In this study, physiological and gene expression analyses under different light intensities were performed in the seedlings of rubber tree (Hevea brasiliensis) clone GT1. When light intensity increased from 20 to 1000 µmol m−2 s−1, there was no effect on the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), indicating that high light intensity did not damage the structure and function of PSII reaction center. However, the effective photochemical quantum yield of PSII (Y(II)), photochemical quenching coefficient (qP), electron transfer rate (ETR), and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (qL) were increased significantly as the light intensity increased, reached a maximum at 200 µmol m−2 s−1, but decreased from 400 µmol m−2 s−1. These results suggested that the PSII photochemistry showed an optimum performance at 200 µmol m−2 s−1 light intensity. The chlorophyll content was increased along with the increase of light intensity when it was no more than 400 µmol m−2 s−1. Since increasing light intensity caused significant increase in H2O2 content and decreases in the per unit activity of antioxidant enzymes SOD and POD, but the malondialdehyde (MDA) content was preserved at a low level even under high light intensity of 1000 µmol m−2 s−1, suggesting that high light irradiation did not induce membrane lipid peroxidation in rubber tree. Moreover, expressions of antioxidant-related genes were significantly up-regulated with the increase of light intensity. They reached the maximum expression at 400 µmol m−2 s−1, but decreased at 1000 µmol m−2 s−1. In conclusion, rubber tree could endure strong light irradiation via a specific mechanism. Adaptation to high light intensity is a complex process by regulating antioxidant enzymes activities, chloroplast formation, and
Price, Edwin R; Brun, Antonio; Fasulo, Verónica; Karasov, William H; Caviedes-Vidal, Enrique
Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75-180 Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (L-proline, MW 115 Da, and D-glucose, MW 180 Da) and two carbohydrates that have no mediated transport (L-arabinose, MW 150 Da, and lactulose, MW 342 Da). Absorption of lactulose (0.61±0.06 nmol min(-1) cm(-1)) was significantly lower than that of the smaller arabinose (1.09±0.04 nmol min(-1) cm(-1)). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10mM and 75 mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1±3.0% to 66.2±7.8% of proline absorption is not transporter-mediated (varying proline from 1 mM to 75 mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.
Fasulo, Verónica; Zhang, ZhiQiang; Chediack, Juan G; Cid, Fabricio D; Karasov, William H; Caviedes-Vidal, Enrique
Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems greater in fliers than in nonfliers, although that conclusion rests mainly on a comparison of flying birds and nonflying mammals because only two frugivorous bat species have been studied. Furthermore, the bats studied so far were relatively large (>85 g, compared with most bat species which are <20 g) and were not insectivores (like about 70 % of bat species). We studied the small (11 g) insectivorous bat Tadarida brasiliensis and tested the prediction that the capacity for paracellular absorption would be as high as in the other bat and avian species studied so far, well above that in terrestrial, nonflying mammals. Using standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption = f) of inert carbohydrate probes: L-arabinose (MM = 150.13) absorbed exclusively by paracellular route and 3OMD-glucose (MM = 194) absorbed both paracellularly and transcellularly. As predicted, the capacity of paracellular absorption in this insectivorous bat was high (L-arabinose f = 1.03 ± 0.14) as in other frugivorous bats and small birds. Absorption of 3OMD-glucose was also complete (f = 1.09 ± 0.17), but >80 % was accounted for by paracellular absorption. We conclude that passive paracellular absorption of molecules of the size of amino acids and glucose is extensive in this bat and, generally in bats, significantly higher than that in nonflying mammals, although the exact extent can be somewhat lower or higher depending on molecule size, polarity and charge.
Farid, Ayman Samir; Jimi, Fumiko; Inagaki-Ohara, Kyoko; Horii, Yoichiro
It is known that hypersensitivity reactions in the gastrointestinal tract, which are primarily mediated by mast cells, are associated with a secretory response of the epithelium and often increased permeability to macromolecules. Studies to date have not examined the effects of hyperpermeability on the absorption of toxic substances normally present in the intestinal lumen such as bacterial LPS. In the present study, we observed that Strongyloides venezuelensis infection in mice decreases the mRNA expression of intestinal epithelial cell junctional molecules (occludin and zonula occludens 1) and increases portal endotoxin levels 4 h after intragastric administration of LPS (20 mg/kg body weight). Furthermore, an increase in the flux of immunoglobulin G into the intestinal lumen was observed 10 days postinfection (PI). An increased rate of LPS absorption was also seen in mice infected with Nippostrongylus brasiliensis on day 14 PI and rats concurrently infected with S. venezuelensis and N. brasiliensis on day 20 PI. On the other hand, infection with Eimeria vermiformis and Eimeria pragensis was not observed to enhance LPS absorption 4 h after intragastric administration of LPS (20 mg/kg body weight), although E. vermiformis infection did inhibit the epithelial cell mRNA expression of zonula occludens 1, but not occludin, on day 9 PI, resulting in a reduced immunoglobulin G flux than that produced by S. venezuelensis infection. Our results suggest that mastocytosis accompanying intestinal nematode infection increases the intestinal absorption of LPS into the portal circulation by suppressing the expression of tight junction molecules.
Zhiyi, Nie; Guijuan, Kang; Yu, Li; Longjun, Dai; Rizhong, Zeng
The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis.
The new combination of Ganaspis brasiliensis (Ihering, 1905) is proposed, and the species is redescribed from historical specimens taken in the Neotropical Region as well as more recent specimens reared from Drosophila suzukii Matsumura, 1931 in South Korea. Drosophila suzukii, otherwise known as th...
Oliveira, M R; Silva, N B; Yamamoto, M E; Chellappa, S
The gonad development and reproductive aspects of Hemiramphus brasiliensis from the coastal waters of Rio Grande do Norte, Brazil were verified. This paper presents data on the length-weight relationship, sex ratio, length at first sexual maturity, gonad development, reproductive period and fecundity of H. brasiliensis. Females of this species predominated in the sampled population and were larger in size than the males. The length at the first sexual maturation of males was 20.8 cm and that of females was 21.5 cm. The macroscopic characteristics of the gonads indicated four maturation stages. Histological studies of gonads of H. brasiliensis showed six phases of oocyte development and four phases of spermatocyte development. The batch fecundity of this species is 1153 (± 258.22) oocytes for 50 g body weight of female. The microscopic characteristics of gonad development indicate that H. brasiliensis is a multiple spawner, with active reproductive period during the months of January to June and October. The reproductive period of this species is independent of the rainy period of the region.
Munhae, Catarina De Bortoli; Morini, Maria Santina De Castro; Bueno, Odair Correa
The association between ants and mealybugs can result in damage to agriculture, including vineyards. In southern Brazil, the ant Linepithema micans F. contributes to the dispersal of Eurhizococcus brasiliensis (Wille) (ground pearl), a root mealybug that can lead to economic losses. In this study, the ant communities in vineyards that were infested or uninfested with ground pearls were evaluated in the primary municipalities that produce the Niágara Rosada variety of grapes in southeastern Brazil. The hypothesis of this study was that the composition of the ant community differs between vineyards with and without E. brasiliensis. The ants were collected using subterranean traps in 10 vineyards infested with this mealybug and 10 uninfested vineyards. There was no significant association between ground pearls and the composition or richness of the ant species. Solenopsis invicta (Buren) (Hymenoptera: Formicidae) was the most frequently observed, and Pheidole aberrans (Mayr), Pheidole subarmata (Mayr), and Brachymyrmex incisus F. were common, especially in the rainy season when ground-pearl nymphs were prevalent in the state of São Paulo. Species from preserved or specialized environments were recorded in the vineyards, even with the use of conventional management techniques.
Espinoza-González, Nelly Alejandra; Welsh, Oliverio; de Torres, Noemi Waksman; Cavazos-Rocha, Norma; Ocampo-Candiani, Jorge; Said-Fernandez, Salvador; Lozano-Garza, Gerardo; Choi, Sung-Hak; Vera-Cabrera, Lucio
Two recently synthesized oxazolidinones: (R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-5-hydroxymethyloxazolidin-2-one (DA-7157) and its corresponding pro-drug (R)-3-(4-(2-(2-methyltetrazol-5-yl)-pyridin-5-yl)-3-fluorophenyl)-2-oxo-5-oxazolidinyl) methyl disodium phosphate (DA-7218), have shown very good activity against several Gram positive bacteria, including Nocardia and Mycobacterium. In the present work we evaluated the therapeutic in vivo effects of DA-7218 on Nocardia brasiliensis. We first determined the plasma concentration of the prodrug in BALB/c mice using several doses and then tested its activity in an in vivo experimental actinomycetoma murine model. At the end of treatment, there was a statistically significant difference between the three drug receiving groups (25, 12.5 and 5 mg/kg) and the control group(saline solution) (p=0.001), proving that DA-7218 is effective for the treatment of experimental murine actinomycetoma. This compound could be a potential option for patients affected with mycetoma by Nocardia brasiliensis.
Li, Hui-Liang; Wei, Li-Ran; Guo, Dong; Wang, Ying; Zhu, Jia-Hong; Chen, Xiong-Ting; Peng, Shi-Qing
In plants MADS-box transcription factors (TFs) play important roles in growth and development. However, no plant MADS-box gene has been identified to have a function related to secondary metabolites regulation. Here, a MADS-box TF gene, designated as HbMADS4, was isolated from Hevea brasiliensis by the yeast one-hybrid experiment to screen the latex cDNA library using the promoter of the gene encoding H. brasiliensis small rubber particle protein (HbSRPP) as bait. HbMADS4 was 984-bp containing 633-bp open reading frame encoding a deduced protein of 230 amino acid residues with a typical conserved MADS-box motif at the N terminus. HbMADS4 was preferentially expressed in the latex, but little expression was detected in the leaves, flowers, and roots. Its expression was inducible by methyl jasmonate and ethylene. Furthermore, transient over-expression and over-expression of HbMADS4 in transgenic tobacco plants significantly suppressed the activity of the HbSRP promoter. Altogether, it is proposed that HbMADS4 is a negative regulator of HbSRPP which participates in the biosynthesis of natural rubber. PMID:27895659
Tan, Wen-Si; Yunos, Nina Yusrina Muhamad; Tan, Pui-Wan; Mohamad, Nur Izzati; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan
N-acylhomoserine lactones (AHL) plays roles as signal molecules in quorum sensing (QS) in most Gram-negative bacteria. QS regulates various physiological activities in relation with population density and concentration of signal molecules. With the aim of isolating marine water-borne bacteria that possess QS properties, we report here the preliminary screening of marine bacteria for AHL production using Chromobacterium violaceum CV026 as the AHL biosensor. Strain T33 was isolated based on preliminary AHL screening and further identified by using 16S rDNA sequence analysis as a member of the genus Vibrio closely related to Vibrio brasiliensis. The isolated Vibrio sp. strain T33 was confirmed to produce N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10 HSL) through high resolution tandem mass spectrometry analysis. We demonstrated that this isolate formed biofilms which could be inhibited by catechin. To the best of our knowledge, this is the first report that documents the production of these AHLs by Vibrio brasiliensis strain T33.
Reichard, Jonathan D; Fellows, Spenser R; Frank, Alexander J; Kunz, Thomas H
Bat wings are important for thermoregulation, but their role in heat balance during flight is largely unknown. More than 80% of the energy consumed during flight generates heat as a by-product, and thus it is expected that bat wings should dissipate large amounts of heat to prevent hyperthermia. We measured rectal (T(r)) and surface (T(s)) temperatures of Brazilian free-tailed bats (Tadarida brasiliensis) as they emerged from and returned to their daytime roosts and calculated sensible heat transfer for different body regions (head, body, wings, and tail membrane). Bats' T(r) decreased from 36.8°C during emergence flights to 34.4°C during returns, and T(s) scaled positively with ambient temperature (T(a)). Total radiative heat loss from bats was significantly greater for a radiative sink to the night sky than for a sink with temperature equal to T(a). We found that free-ranging Brazilian free-tailed bats, on average, do not dissipate heat from their wings by convection but instead dissipate radiative heat (L) to the cloudless night sky during flight ([Formula: see text] W). However, within the range of T(a) measured in this study, T. brasiliensis experienced net heat loss between evening emergence and return flights. Regional hypothermia reduces heat loss from wings that are exposed to potentially high convective fluxes. Additional research is needed to establish the role of wings in evaporative cooling during flight in bats.
Aisenberg, Anita; Baruffaldi, Luciana; González, Macarena
The use of chemical signals in a sexual context is widespread in the animal kingdom. Most studies in spiders report the use of female pheromones that attract potential sexual partners. Allocosa brasiliensis and Allocosa alticeps are two burrowing wolf spiders that show sex-role reversal. Females locate male burrows and initiate courtship before males perform any detectable visual or vibratory signal. So, females of these species would be detecting chemical or mechanical cues left by males. Our objective was to explore the potential for male pheromones to play a role in mate detection in A. brasiliensis and A. alticeps. We designed two experiments. In Experiment 1, we tested the occurrence of male contact pheromones by evaluating female courtship when exposed to empty burrows constructed by males or females (control). In Experiment 2, we tested the existence of male volatile pheromones by evaluating female behaviour when exposed to artificial burrows connected to tubes containing males, females or empty tubes (control). Our results suggest the occurrence of male volatile pheromones that trigger female courtship in both Allocosa species. The sex-role reversal postulated for these wolf spiders could be driving the consequent reversal in typical pheromone-emitter and detector roles expected for spiders.
Romo de Vivar Martínez, Paula Rosario; Bento Soares, Marina
Clevosaurus was a cosmopolitan rhynchocephalian genus, known from the Late Triassic to the Early Jurassic. In South America this genus is represented by C. brasiliensis, an important component of the Linha São Luiz taphocoenosis, on the top of the Norian Santa Maria 2 Sequence of Southern Brazil. The best preserved and most abundant bone elements of C. brasiliensis are dentaries, in which variations of shape and size are observed. The aim of this study is to describe and evaluate the variation, using geometric morphometrics methods. Geometric morphometric analysis of 10 specimens highlights variations in relative size of the dentary. Most of the variation observed for PC1 (83.3%) is likely related to ontogeny, and PC2 (10.0%) is likely related to taphonomic signatures. The development patterns observed, such as the growth of the dentary, consists of differential growth in length between the posterior portion of the dentary, that grows at a higher rate, regarding the anterior portion of the element. This allometric growth is similar to what is observed in other rhynchocephalians and is accompanied by the allometric skull growth, similar to the trend exhibited by clevosaurs. The taphocoenosis is bimodal (juveniles and adults) with a bias towards adult preservation. Some diagenetic influence is reflected in deformed skulls and this is observed in the tangent-plot. Finally, a strong correlation was detected between the taphonomic signatures and the PC2, regarding specially disarticulation and degree of fragmentation. PMID:25793754
Aoki, Yuichi; Takahashi, Seiji; Takayama, Daisuke; Ogata, Yoshiyuki; Sakurai, Nozomu; Suzuki, Hideyuki; Asawatreratanakul, Kasem; Wititsuwannakul, Dhirayos; Wititsuwannakul, Rapepun; Shibata, Daisuke; Koyama, Tanetoshi; Nakayama, Toru
Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H. brasiliensis latex, resulting in 4325 expressed sequence tags (ESTs) assembled into 1308 unigenes (692 contigs and 617 singletons). Quantitative analyses of the transcription levels of high redundancy clones in the ESTs revealed genes highly and predominantly expressed in laticifers, such as Rubber Elongation Factor (REF), Small Rubber Particle Protein and putative protease inhibitor proteins. HRT1 and HRT2, cis-prenyltransferases involved in rubber biosynthesis, was also expressed predominantly in laticifers, although these transcript levels were 80-fold lower than that of REF. The 5'-upstream regions of these laticifer-specific genes were cloned and analyzed in silico, revealing seven common motifs consisting of eight bases. Furthermore, transcription factors specifically expressed in laticifers were also identified. The common motifs in the laticifer-specific genes and the laticifer-specific transcription factors are potentially involved in the regulation of gene expression in laticifers.
Garcia Diaz, Virginia; Aisenberg, Anita; Peretti, Alfredo V
Traditional studies on sexual communication have focused on the exchange of signals during courtship. However, communication between the sexes can also occur during or after copulation. Allocosa brasiliensis is a wolf spider that shows a reversal in typical sex roles and of the usual sexual size dimorphism expected for spiders. Females are smaller than males and they are the roving sex that initiates courtship. Occasional previous observations suggested that females performed body shaking behaviors during copulation. Our objective was to analyze if female body shaking is associated with male copulatory behavior in A. brasiliensis, and determine if this female behavior has a communicatory function in this species. For that purpose, we performed fine-scaled analysis of fifteen copulations under laboratory conditions. We video-recorded all the trials and looked for associations between female and male copulatory behaviors. The significant difference between the time before and after female shaking, in favor of the subsequent ejaculation is analyzed. We discuss if shaking could be acting as a signal to accelerate and motivate palpal insertion and ejaculation, and/or inhibiting male cannibalistic tendencies in this species.
Lopes, Elinia Medeiros; Ventura, Carlos Renato Rezende
We describe the development and juvenile morphology of the sea star Echinaster (Othilia) brasiliensis in order to explore evolutionary developmental modes and skeletal homologies. This species produces large, buoyant eggs (0.6 ± 0.03 mm diameter), and has a typical lecithotrophic brachiolaria larva. The planktonic brachiolaria larva is formed 2-4 days after fertilization, when cilia cover the surface. Early juveniles are completely formed by 18 days of age. Initial growth is supported by maternal nutrients while the stomach continues to develop until 60 days after fertilization, when juveniles reach about 0.5 mm of radius length. The madreporite was observed 88 days after fertilization. In the youngest juvenile skeleton of E. (O.) brasiliensis, the madreporite and odontophore are homologous to those of other recent, non-paxillosid asteroids, and follow the Late Madreporic Mode. The emergence of plates related to the ambulacral system follows the Ocular Plate Rule. The development and juvenile skeletal morphology of this species are similar to those of the few other studied species in the genus Echinaster. This study corroborates the notion that the mode of development--including a short-lived lecithotrophic brachiolaria larva--in all Echinaster species shares a similar pattern that may be conserved throughout the evolutionary history of the group.
Aisenberg, Anita; Baruffaldi, Luciana; González, Macarena
The use of chemical signals in a sexual context is widespread in the animal kingdom. Most studies in spiders report the use of female pheromones that attract potential sexual partners.