RESUMO
Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.
Assuntos
Criptococose , Cryptococcus neoformans , Camundongos , Humanos , Animais , Cryptococcus neoformans/genética , Histonas , Higromicina B , Interações Hospedeiro-Patógeno , Neomicina , BiologiaRESUMO
Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Brazil. The disease is caused by dimorphic fungi nested within the Paracoccidioides genus. We described 106 PCM cases (47.1 cases/year) at the Tropical Diseases Public Hospital of Tocantins State. PCM was prevalent in males and rural workers over 50 years; the chronic pulmonary form predominated in 67% of cases. The male-to-female ratio was 2.65:1, with more women affected than other endemic regions of Brazil. Urban or indoor activities were reported in women and are ascribed to disease urbanization. qPCR-based assays confirmed the identification of Paracoccidioides DNA in 37 biological specimens. Paracoccidioides sp. DNA was found in 53% of the environmental samples, suggesting autochthonous infections. Therefore, the Tocantins-Araguaia basin must be considered a novel hyperendemic area of PCM in Brazil, reinforcing the importance of including PCM as a notifiable disease, requiring specific diagnosis and health measures.
RESUMO
Ergosterol is the most important membrane sterol in fungal cells and a component not found in the membranes of human cells. We identified the ERG6 gene in the AIDS-associated fungal pathogen, Cryptococcus neoformans, encoding the sterol C-24 methyltransferase of fungal ergosterol biosynthesis. In this work, we have explored its relationship with high-temperature growth and virulence of C. neoformans by the construction of a loss-of-function mutant. In contrast to other genes involved in ergosterol biosynthesis, C. neoformans ERG6 is not essential for growth under permissive conditions in vitro. However, the erg6 mutant displayed impaired thermotolerance and increased susceptibility to osmotic and oxidative stress, as well as to different antifungal drugs. Total lipid analysis demonstrated a decrease in the erg6Δ strain membrane ergosterol content. In addition, this mutant strain was avirulent in an invertebrate model of C. neoformans infection. C. neoformans Erg6 was cyto-localized in the endoplasmic reticulum and Golgi complex. Our results demonstrate that Erg6 is crucial for growth at high temperature and virulence, likely due to its effects on C. neoformans membrane integrity and dynamics. These pathogen-focused investigations into ergosterol biosynthetic pathway components reinforce the multiple roles of ergosterol in the response of diverse fungal species to alterations in the environment, especially that of the infected host. These studies open perspectives to understand the participation of ergosterol in mechanism of resistance to azole and polyene drugs. Observed synergistic growth defects with co-inhibition of Erg6 and other components of the ergosterol biosynthesis pathway suggests novel approaches to treatment in human fungal infections.
Assuntos
Criptococose/genética , Cryptococcus neoformans/genética , Ergosterol/biossíntese , Metiltransferases/genética , Antifúngicos/farmacologia , Azóis/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Retículo Endoplasmático/efeitos dos fármacos , Ergosterol/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação/efeitos dos fármacos , Virulência/genéticaRESUMO
Fungal Candida species are commensals present in the mammalian skin and mucous membranes. Candida spp. are capable of breaching the epithelial barrier of immunocompromised patients with neutrophil and cell-mediated immune dysfunctions and can also disseminate to multiple organs through the bloodstream. Here we examined the action of innate defense regulator 1018 (IDR-1018), a 12-amino-acid-residue peptide derived from bovine bactenecin (Bac2A): IDR-1018 showed weak antifungal and antibiofilm activity against a Candida albicans laboratory strain (ATCC 10231) and a clinical isolate (CI) (MICs of 32 and 64 µg · ml-1, respectively), while 8-fold lower concentrations led to dissolution of the fungal cells from preformed biofilms. IDR-1018 at 128 µg · ml-1 was not hemolytic when tested against murine red blood cells and also has not shown a cytotoxic effect on murine monocyte RAW 264.7 and primary murine macrophage cells at the tested concentrations. IDR-1018 modulated the cytokine profile during challenge of murine bone marrow-derived macrophages with heat-killed C. albicans (HKCA) antigens by increasing monocyte chemoattractant protein 1 (MCP-1) and interleukin-10 (IL-10) levels, while suppressing tumor necrosis factor alpha (TNF-α), IL-1ß, IL-6, and IL-12 levels. Mice treated with IDR-1018 at 10 mg · kg-1 of body weight had an increased survival rate in the candidemia model compared with phosphate-buffered saline (PBS)-treated mice, together with a diminished kidney fungal burden. Thus, IDR-1018 was able to protect against murine experimental candidemia and has the potential as an adjunctive therapy.
Assuntos
Antifúngicos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candidemia/tratamento farmacológico , Candidemia/prevenção & controle , Fatores Imunológicos/uso terapêutico , Animais , Candida albicans/imunologia , Candida albicans/isolamento & purificação , Linhagem Celular , Quimiocina CCL2/imunologia , Modelos Animais de Doenças , Interleucina-10/imunologia , Subunidade p35 da Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Vacinas Fúngicas/imunologia , Paracoccidioidomicose/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Vacinas Fúngicas/genética , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Camundongos , Óxido Nítrico/metabolismo , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/prevenção & controle , Paracoccidioidomicose/terapia , Plasmídeos/genética , Baço/imunologia , Baço/metabolismo , Baço/patologia , Vacinas de DNA/genéticaRESUMO
The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.
Assuntos
Antígenos CD/genética , Predisposição Genética para Doença , Paracoccidioidomicose/genética , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Adulto , Idoso , Antígeno CTLA-4 , Estudos de Casos e Controles , Doença Crônica , Feminino , Frequência do Gene , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Itraconazole (ITZ) is a drug used to treat various fungal infections and may cause side effects. The aim of this study was to develop and evaluate the in vitro activity of DMSA-PLGA nanoparticles loaded with ITZ against Paracoccidioides brasiliensis, as well as their cytotoxicity. Nanoparticles were prepared using the emulsification-evaporation technique and characterized by their encapsulation efficiency, morphology (TEM), size (Nanosight) and charge (zeta potential). Antifungal efficacy in P. brasiliensis was determined by minimal inhibition concentration (MIC), and cytotoxicity using MTT assay. ITZ was effectively incorporated in the PLGA-DMSA nanoparticles with a loading efficiency of 72.8 +/- 3.50%. The shape was round with a solid polymeric structure, and a size distribution of 174 +/- 86 nm (Average +/- SD). The particles were negatively charged. ITZ-NANO presented antifungal inhibition (MIC = 6.25 ug/mL) against P. brasiliensis and showed lower in vitro cytotoxicity than free drug (ITZ).
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Itraconazol/administração & dosagem , Itraconazol/toxicidade , Ácido Láctico/química , Nanocápsulas/química , Paracoccidioides/efeitos dos fármacos , Ácido Poliglicólico/química , Succímero/química , Animais , Antifúngicos/administração & dosagem , Antifúngicos/química , Antifúngicos/toxicidade , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Itraconazol/química , Camundongos , Nanocápsulas/ultraestrutura , Paracoccidioides/citologia , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.
Assuntos
Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antígenos CD , Predisposição Genética para Doença , Paracoccidioidomicose , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Doença Crônica , Frequência do Gene , Genótipo , HaplótiposRESUMO
BACKGROUND: The prevalence of invasive fungal infections (IFIs) has increased steadily worldwide in the last few decades. Particularly, there has been a global rise in the number of infections among immunosuppressed people. These patients present severe clinical forms of the infections, which are commonly fatal, and they are more susceptible to opportunistic fungal infections than non-immunocompromised people. IFIs have historically been associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies, including side effects, toxicities, drug interactions and antifungal resistance. Thus, the search for alternative therapies and/or the development of more specific drugs is a challenge that needs to be met. Genomics has created new ways of examining genes, which open new strategies for drug development and control of human diseases. RESULTS: In silico analyses and manual mining selected initially 57 potential drug targets, based on 55 genes experimentally confirmed as essential for Candida albicans or Aspergillus fumigatus and other 2 genes (kre2 and erg6) relevant for fungal survival within the host. Orthologs for those 57 potential targets were also identified in eight human fungal pathogens (C. albicans, A. fumigatus, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Paracoccidioides lutzii, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum). Of those, 10 genes were present in all pathogenic fungi analyzed and absent in the human genome. We focused on four candidates: trr1 that encodes for thioredoxin reductase, rim8 that encodes for a protein involved in the proteolytic activation of a transcriptional factor in response to alkaline pH, kre2 that encodes for α-1,2-mannosyltransferase and erg6 that encodes for Δ(24)-sterol C-methyltransferase. CONCLUSIONS: Our data show that the comparative genomics analysis of eight fungal pathogens enabled the identification of four new potential drug targets. The preferred profile for fungal targets includes proteins conserved among fungi, but absent in the human genome. These characteristics potentially minimize toxic side effects exerted by pharmacological inhibition of the cellular targets. From this first step of post-genomic analysis, we obtained information relevant to future new drug development.
Assuntos
Proteínas Fúngicas/genética , Genômica/métodos , Sequência de Aminoácidos , Aspergillus fumigatus/classificação , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Candida albicans/classificação , Candida albicans/genética , Candida albicans/patogenicidade , Proteínas Fúngicas/química , Humanos , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The conventional treatment for paracoccidioidomycosis, the most prevalent mycosis in Latin America, involves long periods of therapy resulting in sequels and high frequency of relapses. The search for new alternatives of treatment is necessary. Previously, we have demonstrated that the hsp65 gene from Mycobacterium leprae shows prophylactic effects against murine paracoccidioidomycosis. Here, we tested the DNAhsp65 immunotherapy in BALB/c mice infected with Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. We observed an increase of Th1 cytokines accompanied by a reduction in fungal burden and pulmonary injury. These results provide new prospects for immunotherapy of paracoccidioidomycosis and other mycoses.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Imunoterapia/métodos , Mycobacterium leprae/imunologia , Paracoccidioidomicose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/genética , Paracoccidioides/imunologia , Vacinas de DNA/administração & dosagemRESUMO
Paracoccidioidomycosis (PCM) is a systemic disease endemic to most of Latin America, with greatest impact in rural areas. The taxonomic status of one of the best studied Paracoccidioides isolates (Pb01) as P. brasiliensis remains unresolved due to its genomic differences from the other three previously described phylogenetic species (S1, PS2 and PS3; Carrero et al., 2008. Fungal Genet. Biol. 45, 605). Using the genealogic concordance method of phylogenetic species recognition (GCPSR) via maximum parsimony and Bayesian analysis, we identified a clade of 17 genotypically similar isolates, including Pb01, which are distinct from the S1/PS2/P3 clade. Consistent with GCPSR, this "Pb01-like" group can be considered a new phylogenetic species, since it is strongly supported by all independent and concatenated genealogies. "Pb01-like" species exhibit great sequence and morphological divergence from the S1/PS2/PS3 species clade, and we estimate that these groups last shared a common ancestor approximately 32 million years ago. In addition, recombination analysis revealed independent events inside both main groups suggesting reproductive isolation. Consequently, we recommend the formal description of the "Pb01-like" cluster as the new species Paracoccidioides lutzii, a tribute to Adolpho Lutz, discoverer of P. brasiliensis in 1908.
Assuntos
Evolução Molecular , Especiação Genética , Paracoccidioides/genética , Filogenia , Teorema de Bayes , DNA Fúngico/genética , Marcadores Genéticos , Paracoccidioides/classificação , Polimorfismo Genético , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
OBJECTIVES: The present study reports on the preparation and testing of a desoxycholate amphotericin B (D-AMB) sustained delivery system based on poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) polymeric blends (Nano-D-AMB) aimed at reducing the number of AMB administrations required to treat mycosis. METHODS: BALB/c mice were infected with the yeast Paracoccidioides brasiliensis intravenously to mimic the chronic form of paracoccidioidomycosis. At 30 days post-infection, the animals were treated with Nano-D-AMB [6 mg/kg of encapsulated D-AMB, intraperitoneally (ip), interval of 72 h] or D-AMB (2 mg/kg, ip, interval of 24 h). Drug efficacy was investigated by the fungal burden recovery from tissues. Toxicity was assessed by renal and hepatic biochemical parameters, physical appearance of the animals and haematological investigation. The control groups used were non-infected and the infected mice mock treated with PBS. RESULTS: Nano-D-AMB presented results comparable to free D-AMB, with a marked antifungal efficacy. The Nano-D-AMB-treated group presented lower loss of body weight and absence of stress sign (piloerection and hypotrichosis) observed after D-AMB treatment. No renal [blood urea nitrogen (BUN), creatinine] or hepatic (pyruvic and oxalacetic glutamic transaminases) biochemical abnormalities were found. The micronucleus assay showed no significant differences in both the micronucleus frequency and percentage of polychromatic erythrocytes for Nano-D-AMB, indicating the absence of genotoxicity and cytotoxic effects. CONCLUSIONS: The D-AMB-coated PLGA-DMSA nanoparticle showed antifungal efficacy, fewer undesirable effects and a favourable extended dosing interval. Nano-D-AMB comprises an AMB formulation able to lessen the number of drug administrations. Further studies would elucidate whether Nano-D-AMB would be useful to treat systemic fungal infections such as paracoccidioidomycosis, candidiasis, aspergillosis and cryptococcosis.
Assuntos
Anfotericina B/uso terapêutico , Ácido Desoxicólico/uso terapêutico , Ácido Láctico/uso terapêutico , Nanopartículas/uso terapêutico , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Ácido Poliglicólico/uso terapêutico , Succímero/uso terapêutico , Anfotericina B/administração & dosagem , Anfotericina B/efeitos adversos , Animais , Peso Corporal , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Medula Óssea/fisiologia , Contagem de Colônia Microbiana , Ácido Desoxicólico/administração & dosagem , Ácido Desoxicólico/efeitos adversos , Combinação de Medicamentos , Feminino , Rim/efeitos dos fármacos , Rim/fisiologia , Ácido Láctico/administração & dosagem , Ácido Láctico/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/microbiologia , Fígado/fisiologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/efeitos adversos , Ácido Poliglicólico/administração & dosagem , Ácido Poliglicólico/efeitos adversos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Succímero/administração & dosagem , Succímero/efeitos adversos , Resultado do TratamentoRESUMO
Heat-shock proteins are molecules with extensive data showing their potential as immunomodulators of different types of diseases. The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Chaperoninas/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/prevenção & controle , Vacinas de DNA/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Chaperonina 60 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Paracoccidioidomicose/imunologia , Vacinação , Vacinas de DNA/genéticaRESUMO
Paracoccidioiddes brasiliensis is a thermodimorphic fungus endemic to Latin America, where it causes the most prevalent systemic mycosis, paracoccidioidomycosis (PCM). DNA microarray technology has been used to identify patterns of gene expression when a microbe is confronted with conditions of interest, such as in vitro and/or ex vivo interaction with specific cells. P. brasiliensis is one organism that has benefited from this approach. Even though its genome has not been sequenced yet, much has been discovered from its transcriptome and DNA array analyses. In this review, we will outline the current knowledge in P. brasiliensis transcriptome, with focus on differential expression analysis in vitro and on the discussion of the genes that are controlled during the host-pathogen interaction ex vivo in order to give insights into the pathobiology of this fungus. In vitro experiments enabled the delineation of whole metabolic pathways; the description of differential metabolism between mycelium and yeast cells and of the mainly signaling pathways controlling dimorphism, high temperature growth, thermal and oxidative stress, and virulence/ pathogenicity. Recent ex vivo experiments provided advances on the comprehension of the plasticity of response and indicate that P. brasiliensis is not only able to undergo fast and dramatic expression profile changes but can also discern subtle differences, such as whether it is being attacked by a macrophage or submitted to the bloodstream route conditions.
Assuntos
Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Paracoccidioides , Animais , Proteínas Fúngicas/genética , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Paracoccidioides/patogenicidade , Paracoccidioides/fisiologia , Paracoccidioidomicose/microbiologia , VirulênciaRESUMO
The ascomycete Paracoccidioides brasiliensis is a human pathogen with a broad distribution in Latin America. The infection process of P. brasiliensis is initiated by aerially dispersed mycelia propagules, which differentiate into the yeast parasitic phase in human lungs. Therefore, the transition to yeast is an initial and fundamental step in the infective process. In order to identify and characterize genes involved in P. brasiliensis transition to yeast, which could be potentially associated to early fungal adaptation to the host, expressed sequence tags (ESTs) were examined from a cDNA library, prepared from mycelia ongoing differentiation to yeast cells. In this study, it is presented a screen for a set of genes related to protein synthesis and to protein folding/modification/destination expressed during morphogenesis from mycelium to yeast. Our analysis revealed 43 genes that are induced during the early transition process, when compared to mycelia. In addition, eight novel genes related to those processes were described in the P. brasiliensis transition cDNA library. The types of induced and novel genes in the transition cDNA library highlight some metabolic aspects, such as putative increase in protein synthesis, in protein glycosylation, and in the control of protein folding that seem to be relevant to the fungal transition to the parasitic phase.
Assuntos
Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Morfogênese/fisiologia , Micélio/crescimento & desenvolvimento , Paracoccidioides , Leveduras/citologia , Etiquetas de Sequências Expressas , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Humanos , Micélio/genética , Paracoccidioides/citologia , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/patogenicidade , Biossíntese de Proteínas , Dobramento de Proteína , Transcrição Gênica , Leveduras/genética , Leveduras/metabolismoRESUMO
BACKGROUND: Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target. RESULTS: In this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 microM, respectively. CONCLUSION: The data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Estresse Oxidativo/genética , Paracoccidioides/fisiologia , Sequência de Aminoácidos , Benzoquinonas/farmacologia , Sobrevivência Celular , Dosagem de Genes , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/química , Lactamas Macrocíclicas/farmacologia , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/genética , Paracoccidioides/metabolismo , Alinhamento de SequênciaRESUMO
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.
Assuntos
Catalase/metabolismo , Citocromo-c Peroxidase/metabolismo , Estresse Oxidativo , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/enzimologia , Paracoccidioidomicose/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Antioxidantes/metabolismo , Catalase/genética , Citocromo-c Peroxidase/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , Paracoccidioidomicose/microbiologiaRESUMO
Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes a human systemic mycosis with high incidence in Latin America. Owing to their participation in the control of pathogen morphogenesis, differentiation and virulence, it was decided to characterize ras genes in P. brasiliensis. ras1 and ras2 were identified to be coding for two different proteins with high identity. The ras transcriptional pattern was investigated by reverse transcription PCR (RT-PCR) during mycelium-to-yeast (M-->Y) transition, heat shock at 42 degrees C and after internalization of yeast cells by murine macrophages. Both genes were downregulated inside macrophages and ras1, at 42 degrees C. In contrast, ras genes did not show any transcriptional variation during the M-->Y transition. The fact that Ras proteins are attached to the membrane via farnesylation prompted the use of a farnesyltransferase inhibitor to investigate the importance of this process for vegetative growth and dimorphic transition. Farnesylation blockage interfered with the vegetative growth of yeast cells and stimulated germinative tube production even at 37 degrees C. During Y-->M transition, the inhibitor increased filamentation in a dose-dependent manner, indicating that impaired farnesylation favours the mycelium form of P. brasiliensis. The results suggest that ras genes might have a role in dimorphism, heat shock response and in host-pathogen interaction.
Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Paracoccidioides/citologia , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Fúngico/química , DNA Fúngico/genética , Temperatura Alta , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Filogenia , Prenilação , RNA Fúngico/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Paracoccidioides brasiliensis is an important fungal pathogen. The disease it causes, paracoccidioidomycosis (PCM), ranges from localized pulmonary infection to systemic processes that endanger the life of the patient. Paracoccidioides brasiliensis adhesion to host tissues contributes to its virulence, but we know relatively little about molecules and the molecular mechanisms governing fungal adhesion to mammalian cells. Triosephosphate isomerase (TPI: EC 5.3.1.1) of P. brasiliensis (PbTPI) is a fungal antigen characterized by microsequencing of peptides. The protein, which is predominantly expressed in the yeast parasitic phase, localizes at the cell wall and in the cytoplasmic compartment. TPI and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Collectively, these results suggest that TPI is required for interactions between P. brasiliensis and extracellular matrix molecules such as laminin and that this interaction may play an important role in the fungal adherence and invasion of host cells.
Assuntos
Adesão Celular/fisiologia , Proteínas Fúngicas/fisiologia , Paracoccidioides/enzimologia , Paracoccidioides/fisiologia , Triose-Fosfato Isomerase/fisiologia , Animais , Anticorpos Antifúngicos/imunologia , Linhagem Celular , Parede Celular/química , Chlorocebus aethiops , Citoplasma/química , Células Epiteliais/microbiologia , Humanos , Laminina/metabolismo , Microscopia Imunoeletrônica , Ligação ProteicaRESUMO
Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.