RESUMO
Feline-transmitted sporotrichosis has garnered attention due to the recent high incidence and the lack of efficient control in the epicenter of the epidemic, Rio de Janeiro, Brazil. Sporothrix brasiliensis is the major pathogen involved in feline-to-human sporotrichosis in Brazil and displays more virulent genotypes than the closely related species S. schenckii. Over the last two decades, several reports of antifungal-resistant strains have emerged. Sequencing and comparison analysis of the outbreak strains allowed us to observe that the azole non-wild-type S. brasiliensis strain CFP 1054 had significant chromosomal variations compared to wild-type strains. One of these variants includes a region of 231 Kb containing 75 duplicated genes, which were overrepresented for lipid and isoprenoid metabolism. We also identified an additional strain (CFP 1055) that was resistant to itraconazole and amphotericin B, which had a single nucleotide polymorphism in the tac1 gene. The patients infected with these two strains showed protracted clinical course and sequelae. Even though our sample size is modest, these results suggest the possibility of identifying specific point mutations and large chromosomal duplications potentially associated with antifungal resistance and clinical outcomes of sporotrichosis.
Assuntos
Sporothrix , Esporotricose , Animais , Gatos , Humanos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Brasil/epidemiologia , Variações do Número de Cópias de DNA , Polimorfismo de Nucleotídeo Único , Sporothrix/genética , Esporotricose/epidemiologia , Esporotricose/microbiologia , Farmacorresistência Fúngica/genéticaRESUMO
In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.
Assuntos
Coagulação Sanguínea , COVID-19/sangue , Tromboelastografia , Trombofilia/sangue , Adulto , Idoso , Algoritmos , COVID-19/complicações , COVID-19/diagnóstico , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Trombofilia/diagnóstico , Trombofilia/etiologia , Adulto JovemRESUMO
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
Proinflammatory responses are associated with the severity of cerebral malaria. NO, H2O2, eicosanoid and PPAR-γ are involved in proinflammatory responses, but regulation of these factors is unclear in malaria. This work aimed to compare the expression of eicosanoid-forming-enzymes in cerebral malaria-susceptible CBA and C57BL/6 and -resistant BALB/c mice. Mice were infected with Plasmodium berghei ANKA, and the survival rates and parasitemia curves were assessed. On the sixth day post-infection, cyclooxygenase-2 and 5-lipoxygenase in brain sections were assessed by immunohistochemistry, and, NO, H2O2, lipid bodies, and PPAR-γ expression were assessed in peritoneal macrophages. The C57BL/6 had more severe disease with a lower survival time, higher parasitemia and lower production of plasmodicidal NO and H2O2 molecules than BALB/c. Enhanced COX-2 and 5-LOX expression were observed in brain tissue cells and vessels from C57BL/6 mice, and these mice expressed higher constitutive PPAR-γ levels. There was no translocation of PPAR-γ from cytoplasm to nucleus in macrophages from these mice. CBA mice had enhanced COX-2 expression in brain tissue cells and vessels and also lacked PPAR-γ cytoplasm-to-nucleus translocation. The resistant BALB/c mice presented higher survival time, lower parasitemia and higher NO and H2O2 production on the sixth day post-infection. These mice did not express either COX-2 or 5-LOX in brain tissue cells and vessels. Our data showed that besides the high parasite burden and lack of microbicidal molecules, an imbalance with high COX-2 and 5-LOX eicosanoid expression and a lack of regulatory PPAR-γ cytoplasm-to-nucleus translocation in macrophages were observed in mice that develop cerebral malaria.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ciclo-Oxigenase 2/metabolismo , Suscetibilidade a Doenças , Gotículas Lipídicas/metabolismo , Malária Cerebral/metabolismo , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Araquidonato 5-Lipoxigenase/genética , Encéfalo/metabolismo , Encéfalo/parasitologia , Encéfalo/patologia , Ciclo-Oxigenase 2/genética , Expressão Gênica , Macrófagos Peritoneais/metabolismo , Malária Cerebral/mortalidade , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Microglia/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Plasmodium berghei , Transporte ProteicoRESUMO
The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 µM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals.
RESUMO
Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects.
Assuntos
Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Animais , Ácido Butírico/metabolismo , Divisão Celular/efeitos dos fármacos , Cryptococcus neoformans/crescimento & desenvolvimento , Modelos Animais de Doenças , Cápsulas Fúngicas/efeitos dos fármacos , Cápsulas Fúngicas/metabolismo , Ácidos Hidroxâmicos/metabolismo , Lepidópteros , Melaninas/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Fenótipo , Fosfolipases/análise , Análise de Sobrevida , Temperatura , Virulência/efeitos dos fármacosRESUMO
BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.
Assuntos
Doenças do Gato/microbiologia , Proteínas Fúngicas/genética , Sporothrix/genética , Esporotricose/transmissão , Fatores de Virulência/genética , Adaptação Biológica , Animais , Doenças do Gato/transmissão , Gatos , Evolução Molecular , Especiação Genética , Genoma Mitocondrial , Humanos , Filogenia , Sporothrix/classificação , Sporothrix/patogenicidade , Esporotricose/microbiologia , Esporotricose/veterináriaRESUMO
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
BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.
Assuntos
Regulação Fúngica da Expressão Gênica/genética , Micélio/genética , Paracoccidioides/genética , Leveduras/genética , Transporte Biológico/genética , Northern Blotting/métodos , Proteínas de Transporte de Cátions/genética , Parede Celular/genética , Parede Celular/metabolismo , Cisteína/biossíntese , Citoesqueleto/genética , Citoesqueleto/metabolismo , Etiquetas de Sequências Expressas , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Íons/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Leveduras/citologia , beta-Glucosidase/genéticaRESUMO
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.
Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologia , Etiquetas de Sequências Expressas , Proteínas Fúngicas/genética , Humanos , Paracoccidioides/genética , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidade , Transcrição GênicaRESUMO
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.