The homeostasis of iron, copper, and zinc in paracoccidioides brasiliensis, cryptococcus neoformans var. Grubii, and cryptococcus gattii: a comparative analysis

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.

Tipagem molecular, perfis de sensibilidade e caracterização de transcritos diferencialmente expressos durante a infecção de Cryptococcus neoformans

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cryptococcosis outbreak in psittacine birds in Brazil

An outbreak of cryptococcosis occurred in a breeding aviary in São Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 μ g/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil. © 2004 ISHAM.

Molecular typing of IberoAmerican Cryptococcus neoformans isolates

A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with Hhal and Sau961 in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates.

Mycoses associated with AIDS in the Third World

Despite advances in diagnosis and treatment, the epidemiological status of the human immunodeficiency virus (HIV) infection is far from under control in most of the developing world. Sub-Saharan Africa, Southeast Asia and India show increased rates of new infections. In Latin America and the Caribbean there were 1.6 million estimated cases of HIV-infected patients at the end of 1997. Fungal diseases have been one of the most relevant diagnoses in relation to the acquired immunodeficiency syndrome (AIDS), Infections due to Candida species and Cryptococcus neoformans var, neoformans are common worldwide. Histoplasma capsulatum, Coccidioides immitis and Penicillium marneffei are important causes of disease in endemic areas. Infection due to Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis are uncommon even where they are endemic. Phaeohyphomycetes, hyalohyphomycetes and zygomycetes are still rare as a cause of disease among AIDS patients, However, agents pertaining to these groups, such as Aspergillus spp., have an increasing incidence. Superficial mycoses due to dermatophytes have special features from epidemiological, clinical and therapeutic points of view.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or internal proteins are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Estudo microbiológico: amostragem de Cryptococcus sp em São José do Rio Preto/SP

Pós-graduação em Microbiologia - IBILCE

Highlights in pathogenic fungal biofilms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo dos mecanismos de resistência e virulência de isolados de Cryptococcus neoformans e Cryptococcus gattii e quantificação de genes de bomba de efluxo pós tratamento com Galatos de Alquila

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from São Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 São Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 mu g mL(-1). For fluconazole, 22% occurred in the range 8-16 mu g mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.

Cryptococcus gattii and Cryptococcus albidus in captive domestic Pigeon (Columba livia)

Cryptococcosis in animals is an important fungal disease caused by the encapsulated yeast Cryptococcus neoformans. This report describes the occurrence of Cryptococcus gattii and Cryptococcus albidus in domestic pigeon (Columba livia), living together with other birds in a breeding center. The animal presented a pinkish, vascularized mass with gelatinous aspect localized subcutaneously under the right lower eyelid, with approximately 2cm in diameter. At microbiological exam it was isolated Cryptococcus gatti from the eyelid mass, lungs and liver, C. albidus from the trachea and both Cryptococcus species from muscle and kidney.

Caracterização do gene ERG11 envolvido na resistência de Cryptococcus neoformans e Cryptococcus gatti as drogas azólicas e identificação de novos alvos a partir de produtos naturais

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo comparativo do acúmulo de massa seca e macronutrientes por plantas de milho var. BR-106 e Brachiaria plantaginea

Foram realizados dois experimentos, em casa de vegetação, no Departamento de Biologia Aplicada à Agropecuária da FCAV-UNESP de Jaboticabal, objetivando-se determinar o acúmulo de massa seca e a distribuição e acúmulo de macronutrientes em plantas de milho, no período de outubro de 2000 a fevereiro de 2001, e de capim-marmelada, no período de setembro de 2003 a fevereiro de 2004. Os estudos foram realizados em delineamento experimental inteiramente casualizado, com quatro repetições. As plantas cresceram em vasos com capacidade de sete litros - preenchidos com areia de rio lavada e peneirada - e foram irrigadas diariamente com solução nutritiva. Os tratamentos foram representados pelas épocas de amostragem realizadas a intervalos de 14 dias, a saber: 21, 35, 49, 63, 77, 91, 105, 119 e 133 dias após a emergência (DAE) das plantas de milho; e 21, 35, 49, 63, 77, 91, 105, 119, 133 e 147 DAE das plantas de capim-marmelada. O ponto de máximo acúmulo teórico de massa seca deu-se aos 122 DAE para o milho (143,8 g por planta) e aos 143 DAE para o capim-marmelada (23,9 g por planta). A taxa de absorção diária dos macronutrientes atingiu maiores valores entre 71 e 104 DAE para o milho e entre 96 e 111 DAE para a planta daninha. Levando-se em conta a média dos valores de pontos de inflexão observados na cultura do milho, aos 85 DAE uma planta de milho acumula, teoricamente, 83,0 g de massa seca; 788,9 mg de N; 137,5 mg de P; 1.385,6 mg de K; 551,8 mg de Ca; 217,9 mg de Mg; e 92,5 mg de S. Enquanto que, no mesmo período, uma planta de B. plantaginea acumula, teoricamente, 9,6 g de massa seca; 127,8 mg de N; 15,9 mg de P; 217,3 mg de K; 43,9 mg de Ca; 58,3 mg de Mg; e 15,8 mg de S.

Influência de genótipos de couve (Brassica oleracea L. var. acephala DC.) na biologia de Plutella xylostella (L., 1758) (Lepidoptera: Plutellidae)

Objetivou-se, com este trabalho, avaliar a influência de alguns genótipos de couve (Brassica oleracea L. var. acephala DC.) no desenvolvimento de Plutella xylostella (L., 1758) (Lepidoptera: Plutellidae). Os genótipos avaliados foram: Manteiga de Ribeirão Pires I-2620, Roxa I-919, Manteiga de São José, Manteiga de Monte Alegre, Pires 2 de Campinas, Couve Comum, Couve de Arthur Nogueira 2, Couve de Arthur Nogueira 1. Lagartas recém-eclodidas foram mantida em discos foliares de 8 cm de diâmetro para cada genótipo. Foram analisados os seguintes parâmetros: duração e viabilidade das fases larval e pupal, longevidade e fecundidade de adultos, utilizando análises paramétricas e de agrupamentos para interpretação dos dados. Observou-se um prolongamento em dias no ciclo de P. xylostella, aumento no peso de pupa e maiores valores de viabilidade e fecundidade, durante a segunda geração. O genótipo Couve de Arthur Nogueira 2 foi menos favorável ao desenvolvimento de P. xylostella nas duas gerações, e Couve Comum demonstrou maior influência negativa ao inseto na segunda geração. Manteiga de Ribeirão Pires I-2620 foi o mais suscetível nas duas gerações, agrupando com este na segunda geração Pires 2 de Campinas e Manteiga de São José.

Efeito de Colletotrichum dematium var. truncata e Cercospora kikuchii na germinação de sementes de soja

A antracnose, causada por Colletotrichum dematium var. truncata, é a principal doença da soja que afeta a fase inicial de formação das vagens, podendo causar a morte das plântulas. Cercospora kikuchii é o fungo causador da doença mancha púrpura nas sementes de soja, responsável por severas reduções no rendimento e na qualidade das sementes. O objetivo deste trabalho foi determinar o tempo mais apropriado para infecção das sementes de soja por C. dematium var. truncata e C. kikuchii, para posterior avaliação dos danos causados pelo fungo na germinação. Tais fungos foram cultivados em meio BDA. As sementes de soja cultivar MSOY 6101 foram colocadas sobre meio contendo o patógeno C. dematium var. truncata por 0 (testemunha), 4, 16, 24, 32 e 40h. As sementes da cv. CD 208 foram inoculadas com C. kikuchii pelos mesmos períodos citados, com adição de 48 e 56h. Após os respectivos tempos de contato, as sementes foram submetidas ao teste de sanidade (papel de filtro), com duas variações, sem e com assepsia superficial (hipoclorito de sódio 1% por três minutos). Determinado o tempo mais adequado de infecção, outras sementes foram infectadas pelo patógeno e, posteriormente, foram realizados testes de germinação em papel e areia com uma mistura de sementes sadias (colocadas sobre o meio BDA) e sementes inoculadas, resultando em lotes com 0, 20, 40, 60, 80 e 100% de sementes infectadas. O tempo de incubação de 40 e 56 horas para C. dematium var truncata e C. Kikuchii, respectivamente são suficientes para obtenção da totalidade das sementes infectadas. C. Kikuchii não prejudica a germinação das sementes.