Strain differentiation of Trichophyton rubrum by random amplification of polymorphic DNA (RAPD)

Trichophyton rubrum é um importante agente causal de dermatomicose. Os métodos de tipagem molecular têm sido recentemente desenvolvidos para responder questões sobre epidemiologia e auxiliar no esclarecimento de recidivas, após o tratamento. As seqüências aleatórias 1- (5'-d[GGTGCGGGAA]-3') e 6- (5'-d[CCCGTCAGCA]-3') foram usadas para tipagem molecular deste fungo por RAPD produzindo variabilidade intraespecífica. Cinco padrões foram observados entre os 10 isolados de T. rubrum, com ambas as seqüências. Foi concluído que a análise por RAPD pode ser utilizada para estudos epidemiológicos.Trichophyton rubrum is an important cause of dermatomycoses. Molecular strain typing methods have recently been developed to address questions about epidemiology and source of relapse following treatment. This report describes the application of RAPD for molecular strain differentiation of this fungus utilizing the primers 1- (5'-d[GGTGCGGGAA]-3') and 6- (5'-d[CCCGTCAGCA]-3'). A total of five RAPD patterns were observed among 10 strains of T. rubrum, with each of the primers used. We conclude that RAPD analysis using primers 1 and 6 can be used in epidemiological studies

Tipagem molecular de Candida albicans isoladas de pacientes hospitalizados

SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD) and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions) and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.RESUMO Introdução: A maioria das infecções fúngicas hospitalares são causadas por Candida spp. e C. albicans é a espécie mais comumente identificada. Métodos moleculares são ferramentas importantes para a avaliação da origem das leveduras isoladas em hospitais. Métodos: Este é um estudo sobre o perfil genético de 39 isolados clínicos nosocomiais de C. albicans através das técnicas de RAPD e microssatélite, foram usados dois diferentes iniciadores para cada técnica. Resultados: RAPD forneceu 10 e 11 diferentes perfis com valores de SAB 0,84 ± 0,126 e 0,88 ± 0,08 para os primers M2 e P4, respectivamente. A análise de microssatélites, usando os marcadores CDC3 e HIS3 permitiu a observação de seis e sete diferentes alelos respectivamente, com poder discriminatório combinado de 0,91. Conclusões: Embora seja clara a variabilidade genética, foi possível identificar alta similaridade, sugerindo origem comum para pelo menos parte deles. É importante enfatizar que foi comprovada origem comum de leveduras isoladas de colonização (urina, cateter ou secreção orotraqueal) e hemocultura do mesmo paciente, indicando que a candidemia deve ter iniciado a partir de um sítio de colonização. A combinação das técnicas RAPD e microssatélites fornece uma análise rápida e eficiente para investigação de similaridade entre isolados nosocomiais de C. albicans

Proteomic Analysis of Paracoccidioides brasiliensis During Infection of Alveolar Macrophages Primed or Not by Interferon-Gamma

Although members of the Paracoccidioides complex are not obligate intracellular pathogens, they present the ability to survive and multiply inside epithelial cells and phagocytes of mammals, which may favor the spread of the fungus in host tissues. Macrophages resident in the lung are the first line of defense against paracoccidioidomycosis (PCM), presenting mechanisms to control the pathogen dissemination through the granuloma formation or eliminating the fungus through phagocytosis. Phagocytosis triggers an oxidative burst, in which there is an increase in the production of toxic elements, derived from oxygen and nitrogen. The interior of the phagolysosome is a harsh environment to the internalized pathogens, since in addition to the oxygen and nitrogen reactive species, microorganisms face nutrient shortages and proteases activity. Through the NanoUPLC-MSE technology, we analyzed the proteomic response of Paracoccidioides brasiliensis during the infection of alveolar macrophages primed or not by interferon gamma (IFN-γ). At 6 hs post-infection, only (IFN-γ)-primed macrophages were able to kill the fungus. We observed the regulation of amino acids degradation, tricarboxylic acid cycle, respiratory chain, ATP synthesis, glyoxylate cycle, as well as an increase in the expression of defense proteins related to oxidative stress, heat shock, and virulence factors under both conditions analyzed. However, some pathways described as essential for the survival of pathogens inside macrophages were observed only or with higher intensity in yeast cells recovered from non-primed macrophages, as phosphate pentoses pathway, methylcitrate cycle, synthesis of cell wall components, and mitochondrial activity. The data indicate that the intracellular environment of non-primed macrophages could be more permissive to the survival and multiplication of P. brasiliensis. The identification of key molecules for the establishment of infection can help the understanding of the nature of the parasite–host relationship and pathogenesis of PCM

Genotipagem, sorotipagem e determinação de mating-type de isolados clínicos de Cryptococcus neoformans do Estado de São Paulo, Brasil

The basidiomycetous yeast Cryptococcus neoformans is an important fungal pathogen mainly in immunocompromised patients. In this study, 47 clinical isolates of C. neoformans from regions of São Paulo State were studied serologically by using the Crypto Check Iatron RM 304-K kit, their genetic diversity was estimated by PCR-fingerprinting with a microsatellite-specific sequence (GACA)4, RAPD with primer 6 (Amersham Pharmacia Biotech), PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) digested with AvaI and mating type analysis by PCR. All 47 strains isolated from HIV positive patients included in this study were serotype A and MATalpha. The majority of the isolates (45/47) were VNI and only two were VNII by PCR-fingerprinting and PCR-RFLP analysis. High degree of homogeneity was observed when (GACA)4 was used, being highly correlated (>; 0.9). In contrast, the RAPD analysis was more heterogeneous with higher number of molecular profiles. By PCR-RFLP, no new molecular type was found, enhancing the suggestion that the differences based on conserved gene as PLB1, can be resultant of ongoing divergent evolution within the C. neoformans complex, into the current eight subtypes. Our results furnish new information on the molecular epidemiology of C. neoformans in the southeast region of Brazil.Cryptococcus neoformans, pertencente à classe dos basidiomicetos, é um importante patógeno, principalmente em pacientes imunocomprometidos. Neste estudo, 47 isolados clínicos de C. neoformans de várias regiões do Estado de São Paulo foram avaliados quanto aos sorotipos e ao mating-type por PCR. A diversidade genética foi analisada por PCR-fingerprinting com a seqüência iniciadora específica para regiões microssatélite (GACA)4, RAPD com o iniciador 6 (Amersham Pharmacia Biotech) e por RFLP do gene PLB1 digerido com AvaI. Todos os isolados foram obtidos de pacientes HIV positivos e identificados como sorotipo A e MATalfa. A maioria dos isolados pertencia ao tipo molecular VNI (45/47) e apenas dois foram VNII quando analisados por PCR-fingerprinting e PCR-RFLP. Homogeneidade alta foi obtida com o iniciador (GACA)4, com a maioria dos isolados apresentando correlação alta (>; 0.9). Os resultados do RAPD, por sua vez, revelaram maior heterogeneidade com número maior de perfis moleculares. Por PCR-RFLP, nenhum tipo molecular novo foi encontrado, realçando a idéia de que em genes conservados como PLB1, as diferenças podem ser resultantes de divergências evolutivas dentro do complexo C. neoformans, separando os isolados nos oito subtipos moleculares já estabelecidos. Nossos resultados fornecem novas informações sobre a epidemiologia molecular de C. neoformans na região sudeste do Brasil

ANTIFUNGAL POTENTIAL OF PLANT SPECIES FROM BRAZILIAN CAATINGA AGAINST DERMATOPHYTES

Trichophyton rubrum and Trichophyton mentagrophytes complex, or Trichophyton spp. are the main etiologic agents of dermatophytosis, whose treatment is limited by the high cost of antifungal treatments, their various side effects, and the emergence of resistance amongst these species. This study evaluated the in vitro antidermatophytic activity of 23 crude extracts from nine plant species of semiarid vegetation (caatinga) found in Brazil. The extracts were tested at concentrations ranging from 1.95 to 1,000.0 mg/mL by broth microdilution assay against the reference strains T. rubrum ATCC 28189 and T. mentagrophytesATCC 11481, and 33 clinical isolates of dermatophytes. All plants showed a fungicidal effect against both fungal species, with MIC/MFC values of the active extracts ranging from 15.6 to 250.0 µg/mL. Selected extracts of Eugenia uniflora (AcE), Libidibia ferrea (AE), and Persea americana (AcE) also exhibited a fungicidal effect against all clinical isolates of T. rubrum and T. mentagrophytes complex. This is the first report of the antifungal activity of Schinus terebinthifolius, Piptadenia colubrina, Parapiptadenia rigida, Mimosa ophthalmocentra, and Persea americana against both dermatophyte species

Comparative Proteomic Analysis of Histoplasma capsulatum Yeast and Mycelium Reveals Differential Metabolic Shifts and Cell Wall Remodeling Processes in the Different Morphotypes

Histoplasma capsulatum is a thermally dimorphic fungus distributed worldwide, but with the highest incidence in the Americas within specific geographic areas, such as the Mississippi River Valley and regions in Latin America. This fungus is the etiologic agent of histoplasmosis, an important life-threatening systemic mycosis. Dimorphism is an important feature for fungal survival in different environments and is related to the virulence of H. capsulatum, and essential to the establishment of infection. Proteomic profiles have made important contributions to the knowledge of metabolism and pathogenicity in several biological models. However, H. capsulatum proteome studies have been underexplored. In the present study, we report the first proteomic comparison between the mycelium and the yeast cells of H. capsulatum. Liquid chromatography coupled to mass spectrometry was used to evaluate the proteomic profile of the two phases of H. capsulatum growth, mycelium, and yeast. In summary, 214 and 225 proteins were only detected/or preferentially abundant in mycelium or yeast cells, respectively. In mycelium, enzymes related to the glycolytic pathway and to the alcoholic fermentation occurred in greater abundance, suggesting a higher use of anaerobic pathways for energy production. In yeast cells, proteins related to the tricarboxylic acid cycle and response to temperature stress were in high abundance. Proteins related to oxidative stress response or involved with cell wall metabolism were identified with differential abundance in both conditions. Proteomic data validation was performed by enzymatic activity determination, Western blot assays, or immunofluorescence microscopy. These experiments corroborated, directly or indirectly, the abundance of isocitrate lyase, 2-methylcitrate synthase, catalase B, and mannosyl-oligosaccharide-1,2-alpha-mannosidase in the mycelium and heat shock protein (HSP) 30, HSP60, glucosamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate deaminase, and N-acetylglucosamine-phosphate mutase in yeast cells. The proteomic profile-associated functional classification analyses of proteins provided new and interesting information regarding the differences in metabolism between the two distinct growth forms of H. capsulatum

Interacting with hemoglobin: Paracoccidioides spp. recruits hsp30 on its cell surface for enhanced ability to use this iron source

Paracoccidioides spp. are thermally dimorphic fungi that cause paracoccidioidomycosis and can affect both immunocompetent and immunocompromised individuals. The infection can lead to moderate or severe illness and death. Paracoccidioides spp. undergo micronutrients deprivation within the host, including iron. To overcome such cellular stress, this genus of fungi responds in multiple ways, such as the utilization of hemoglobin. A glycosylphosphatidylinositol (GPI)-anchored fungal receptor, Rbt5, has the primary role of acquiring the essential nutrient iron from hemoglobin. Conversely, it is not clear if additional proteins participate in the process of using hemoglobin by the fungus. Therefore, in order to investigate changes in the proteomic level of P. lutzii cell wall, we deprived the fungus of iron and then treated those cells with hemoglobin. Deprived iron cells were used as control. Next, we performed cell wall fractionation and the obtained proteins were submitted to nanoUPLC-MSE. Protein expression levels of the cell wall F1 fraction of cells exposed to hemoglobin were compared with the protein expression of the cell wall F1 fraction of iron-deprived cells. Our results showed that P. lutzii exposure to hemoglobin increased the level of adhesins expression by the fungus, according to the proteomic data. We confirmed that the exposure of the fungus to hemoglobin increased its ability to adhere to macrophages by flow cytometry. In addition, we found that HSP30 of P. lutzii is a novel hemoglobin-binding protein and a possible heme oxygenase. In order to investigate the importance of HSP30 in the Paracoccidioides genus, we developed a Paracoccidioides brasiliensis knockdown strain of HSP30 via Agrobacterium tumefaciens-mediated transformation and demonstrated that silencing this gene decreases the ability of P. brasiliensis to use hemoglobin as a nutrient source. Additional studies are needed to establish HSP30 as a virulence factor, which can support the development of new therapeutic and/or diagnostic approaches.We thank CAPES: CNPq and FAPEG for providing fellowships to A.F.d.S, M.V.T., K.S.F.eS., J.S.d.C, C.A.P., L.C.B., J.D.P. and R.A.G., F.R. was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013). C.M.d.A.S. and M.P. belong to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG), Instituto Nacional de Ciência e Tecnologia (INCT) de Estratégias de Interação Patógeno-Hospedeiro (grant number 201810267000022/INCT-FAPEG)

Metabolic Peculiarities of Paracoccidioides brasiliensis Dimorphism as Demonstrated by iTRAQ Labeling Proteomics

Paracoccidioidomycosis (PCM), a systemic mycosis with a high incidence in Latin America, is caused by thermodimorphic fungi of the Paracoccidioides genus. The contact with host occurs by the inhalation of conidia or mycelial propagules which once reaching the pulmonary alveoli differentiate into yeast cells. This transition process is vital in the pathogenesis of PCM allowing the fungus survival in the host. Thus, the present work performed a comparative proteome analysis of mycelia, mycelia-to-yeast transition, and yeast cells of Paracoccidioides brasiliensis. For that, tryptic peptides were labeled with iTRAQ and identified by LC–MS/MS and computational data analysis, which allowed the identification of 312 proteins differentially expressed in different morphological stages. Data showed that P. brasiliensis yeast cells preferentially employ aerobic beta-oxidation and the tricarboxylic acid cycle accompanied by oxidative phosphorylation for ATP production, in comparison to mycelia and the transition from mycelia-to-yeast cells. Furthermore, yeast cells show a metabolic reprogramming in amino acid metabolism and in the induction of virulence determinants and heat shock proteins allowing adaptation to environmental conditions during the increase of the temperature. In opposite of that, the alcoholic fermentation found to P. lutzii, at least under laboratory conditions, is strongly favored in mycelium compared to yeast cells. Thereby, the data strongly support substantial metabolic differences among members of the Paracoccidioides complex, when comparing the saprobiotic mycelia and the yeast parasitic phases