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Previous issue date: 2009-03-31 / Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic
mycosis presenting clinical manifestations ranging from mild to severe forms. A P.
brasiliensis cDNA expression library was produced and screened with pooled sera from
PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis
yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera
indicated 35 open reading frames presenting homology to genes involved in metabolic
pathways, transport, among other predicted functions. The complete cDNAs encoding
aromatic L-amino acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a
homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant
proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The
proteins and the synthetic peptide were recognized by sera of patients with confirmed
PCM and not by sera of healthy patients. Using the vivo-induced antigen technology
(IVIAT) we identified immunogenic proteins expressed at high levels during infection.
Quantitative real - time RT-PCR demonstrated high transcript levels of Pbddc, Pbls and
Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from
spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest
a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. / Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose (PCM),
uma micose sistêmica, prevalente nos países da América Latina. Uma biblioteca de
cDNA de expressão de Paracoccidioides brasiliensis foi construída e rastreada com
soros de pacientes acometidos por paracoccidioidomicose (PCM). Foram identificados
35 clones de cDNAs que codificam proteínas relacionadas com metabolismo celular,
transporte, energia, transcrição, endereçamento de proteínas, transdução de sinal e
componentes celulares. Os cDNAs codificantes da L- aminoacido aromatico -
descarboxilase (Pbddc), da lumazina sintase (Pbls) e do transportador de cobre de alta
afinidade foram obtidos. As proteínas recombinantes PbDDC e PbLS e o peptídio
sintético PbCTR3 foram reconhecidos por soros de pacientes com PCM e não reagiram
com soros controle. A técnica de IVIAT (tecnologia de antígenos induzidos in vivo)
propiciou a identificação de proteínas imunogênicas mais expressas durante o processo
infecçioso. RT-PCR em tempo real quantitativa (qRT-PCR) demostrou altos níveis de
transcritos de Pbddc, Pbls e Pbctr3 em células leveduriformes infectando macrófagos.
O transcritos de células leveduriformes de P. brasiliensis recuperadas de fígado e baço
de camundongos foram medidos por qRT-PCR. Estes resultados sugerem o provável
papel das proteínas imunogênicas no processo infeccioso de P. brasiliensis.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.bc.ufg.br:tde/1588 |
| Date | 31 March 2009 |
| Creators | DANTAS, Sabrina Fonseca Ingênito Moreira |
| Contributors | SOARES, Célia Maria de Almeida |
| Publisher | Universidade Federal de Goiás, Doutorado em Medicina Tropical, UFG, BR, Ciências da Saúde |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFG, instname:Universidade Federal de Goiás, instacron:UFG |
| Rights | info:eu-repo/semantics/openAccess |
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