XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:19-2

Oral / Poster

19-2

Cell surface-associated proteins in the yeast phase of Paracoccidioides brasiliensis: characterization and localization of Prx1p

Authors:

Larissa Valle Guilhen Longo (UNIFESP - Universidade Federal de Sao Paulo) ; Ernesto S. Nakayasu (UTEP - University of Texas at El Paso) ; Milene Carmes Vallejo (UNIFESP - Universidade Federal de Sao Paulo) ; Alisson Leonardo Matsuo (UNIFESP - Universidade Federal de Sao Paulo) ; Tiago José Paschoal Sobreira (INCOR - Heart Institute) ; Luciane Ganiko (UTEP - University of Texas at El Paso) ; Igor Correia Almeida (UTEP - University of Texas at El Paso) ; Rosana Puccia (UNIFESP - Universidade Federal de Sao Paulo)

Abstract

The fungal cell wall is a complex and dynamic structure and the main interface with the host. It consists mainly of polysaccharides (glucans and chitin), but also of lipids and covalently-linked structural (glyco)proteins. Many proteins have recently been individually localized to the cell wall of Paracoccidioides brasiliensis, such as glyceraldehyde-3-phosphate dehydrogenase, enolase, malate synthase, laccase, triosephosphate isomerase, formamidase and Mdj1, which are originally intracellular. In our preliminary proteomic studies of P. brasiliensis surface proteins, the mitochondrial peroxiredoxin Prx1 was detected. In Candida albicans, the Prx1 homologous thiol-specific antioxidant Tsa was differentially localized at the cell wall of the pathogenic hyphae. Presently, we aimed at characterizing Prx1 in P. brasiliensis. The PRX1 gene was cloned, the heterologous protein was expressed in E. coli Bl21 (DE3) plysS and anti-Prx1 antibodies were produced in rabbits. Immunoblots of cell wall dithiothreitol extracts from Pb18 yeasts were tested with serum from a rabbit immunized with the recombinant protein and revealed a band of nearly 25 kDa, consistent with Prx1 predicted molecular mass. Flow citometry experiments showed that in non-permeable Pb18 yeasts anti-Prx1 induced higher fluorescence when compared to the control. In parallel we carried out proteomic analysis of Pb3 cell surface-associated proteins. Yeast cells were cultivated in Hamâ€™s defined medium/glucose in the presence or absence of human plasma (pl) and extensively washed cell-free cell wall samples were prepared. Proteins were extracted with sodium dodecyl sulfate (SDS), digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, 160 proteins with at least 10 spectra were identified in Pb3 and 60 in Pb3pl; they have very distinct functions. Enolase, 14-3-3, superoxide dismutase and Prx1 are among them. They were also detected in extracellular vesicles, which might justify their surface localization. Negative Prx1 modulation by plasma was observed only in Pb18, where a similar study was performed. Further investigation of plasma regulation on Prx1 expression in Pb3 and Pb18 will be carried out, as well as on the role of the protein on the cell surface. Support: FAPESP, CNPq, NIH (grants # 5G12RR008124-16A1 and 5G12RR008124-16A1S1)਀

Keyword: cell wall, Paracoccidioides brasiliensis, peroxiredoxin, Prx1p, thiol-specific antioxidant

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