Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis

Relber Aguiar Gonçales; Igor Godinho Portis; Thaila Fernanda Dos Reis; Luiz Roberto Basso Júnior; Roberto Martinez; Heng Zhu; Maristela Pereira; Célia Maria de Almeida Soares; Paulo Sergio Rodrigues Coelho

doi:10.3389/ffunb.2023.1243475

Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis

Front Fungal Biol. 2023 Aug 29:4:1243475. doi: 10.3389/ffunb.2023.1243475. eCollection 2023.

Authors

Relber Aguiar Gonçales^{1

2}, Igor Godinho Portis³, Thaila Fernanda Dos Reis⁴, Luiz Roberto Basso Júnior⁵, Roberto Martinez⁶, Heng Zhu⁷, Maristela Pereira³, Célia Maria de Almeida Soares³, Paulo Sergio Rodrigues Coelho⁵

Affiliations

¹ Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal.
² ICVS/3B's - PT Government Associate Laboratory, Braga, Portugal.
³ Laboratorio de Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brazil.
⁴ Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.
⁵ Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto Medical School, Universidade de São Paulo (FMRP/USP), Ribeirão Preto, SP, Brazil.
⁶ Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brazil.
⁷ Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, United States.

Abstract

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Keywords: GPI-anchored proteins; PCM; Paracoccidioides brasiliensis; Western blot; cell wall.

Grants and funding

The research conducted at Universidade de São Paulo was aided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). RG received a fellowship from FAPESP (grant: 2014/22561-7) and CAPES (grants: 99999.010699/2014-07, 88887.333726/2019-00), while PC was supported by funding from FAPESP (grant: 08/558316). MP and CS was supported by funding from “FAPEG/PRONEX (Fundação de Apoio à Pesquisa do Estado de Goiás/Programa de Apoio a Núcleos de Excelência)”.