XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:64-1

Oral / Poster

64-1

Characterization of Paracoccidioides brasiliensis prostaglandin as an authentic PGE2

Authors:

Ana Paula Bordon-graciani (UNESP - IB - Universidade Estadual Paulista) ; Guilherme Augusto Biondo (UNESP - IB - Universidade Estadual Paulista) ; Luciane Alarcão Dias-melicio (UNESP - IB - Universidade Estadual Paulista) ; Daniela Ramos Rodrigues (UNESP - IB - Universidade Estadual Paulista) ; Tatiana Fernanda Bachiega (UNESP - IB - Universidade Estadual Paulista) ; Reginaldo Keller Fernandes (UNESP - IB - Universidade Estadual Paulista) ; Cilmery Suemi Kurokawa (UNESP - FMB - Universidade Estadual Paulista) ; João Pessoa Araujo Jr. (UNESP - IB - Universidade Estadual Paulista) ; Edivaldo Domingues Velini (UNESP - FCA - Universidade Estadual Paulista) ; Angela Maria Victoriano de Campos Soares (UNESP - IB - Universidade Estadual Paulista)

Abstract

Introduction and objectives: Prostaglandin E2 (PGE2) is an eicosanoid synthesized from arachidonic acid (AA) by a complex metabolic pathway involving the enzyme cyclooxygenase (COX) in mammalian hosts. Some works have been demonstrated that pathogenic fungi produce authentic PGE2 by action of dioxygenase (linoleate diol synthase) enzyme, which has functional homology to mammalian COX. Thus, the purpose of this work was to conclusively identify the capacity of P. brasiliensis to produce authentic PGE2 and if this production involves dioxygenase enzyme pathway, already identified for other fungi. Methodology: Pb18 and Pb265 yeast cells were suspended in RPMI culture medium and incubated by 4 hours in absence or presence of AA and the PGE2 levels was measured by ELISA using the Prostaglandin E2 High Sensitivity Immunoassay Kit. To confirm the production and molecular structure of PGE2, these cultures were analyzed by HPLC and mass spectrometry (MS). Moreover, on Pb18 and Pb265 yeast cell cultures, the presence of mRNA coding for enzyme dioxygenase, the possible enzyme involved in PGE2 production by P. brasiliensis was investigated by real-time RT-PCR. Results: Both strains of P. brasiliensis produced PGE2 whose levels were increased after AA treatment and inhibited with monoclonal antibody anti-PGE2 incubation. This production was confirmed by HPLC and MS analysis since it has a mass and an elution time identical to those of the commercial PGE2 standard. Real-time RT-PCR assays do not showed expression to COX-1 and COX-2 genes. On the contrary, expression of linoleate diol synthase (dioxygenase) was observed. Conclusions: Our results showed that P. brasiliensis produces PGE2 identical to that of mammalian host by using endogenous or exogenous sources of AA. Moreover, these data suggest that a novel enzyme pathway may exist in this fungus, possibly involving linoleate diol synthase. Finnancial support: FAPESP.

Keyword: authentic PGE2, dioxygenase, HPLC, Mass spectrometry, Paracoccidioides brasiliensis

਀�