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PbGP43 Genotyping Using Paraffin-Embedded Biopsies of Human Paracoccidioidomycosis Reveals a Genetically Distinct Lineage in the Paracoccidioides brasiliensis Complex

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Abstract

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.

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Acknowledgements

We thank Prof. Marcello F. Franco, MD, who was mentor to GR in her Master and PhD program and that through his passion for fungus, mainly Paracoccidioides brasiliensis showed her the importance of the mycology in the world. Prof. Arnaldo L. Colombo, MD (Laboratório Especial de Micologia – LEMI – Disciplina de Infectologia, Departamento de Medicina, UNIFESP) for making part of the molecular analyses performed in this study available and Prof. Zoilo P. Camargo (Disciplina de Biologia Celular, Departamento de Microbiologia, Imunologia e Parasitologia, UNIFESP) for providing Histoplasma capsulatum DNA as a negative control. The authors are also grateful to Prof. Sigrid de Sousa Santos, MD (Departamento de Medicina, Universidade Federal de São Carlos, SP, Brasil) and the following pathologists: Clovis Klock, MD; Eduardo Haruo Suguiama, MD; Fernando Tito Motta, MD; Flavio Lima, MD; Gisela Nascimento Silva, MD; Margarida M. F. Moraes, MD; Paulo Faria,MD; Rogério Mendes Grande, MD for sending paraffin-embedded tissue blocks from PCM patients.

Funding

This work was supported by the grant from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [finance code 001 to G.R. and E.B.C.]; the Conselho Nacional de Desenvolvimento Científico e Tecnológico [Grant No. 313375/2017-8 to R.P.]; and the Fundação de Amparo à Pesquisa do Estado de São Paulo [Grant No. 06-05095-6 to R.P.].

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  1. Giannina Ricci and Emeline Boni Campanini both authors contributed equally.

Authors and Affiliations

  1. Centro de Diagnóstico e Pesquisa em Biologia Molecular Dr Ivo Ricci, São Carlos, SP, Brazil

    Giannina Ricci & Angela Satie Nishikaku

  2. Departamento de Patologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil

    Giannina Ricci

  3. Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Carlos, SP, Brazil

    Emeline Boni Campanini

  4. Disciplina de Biologia Celular, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil

    Rosana Puccia & Anderson Messias Rodrigues

  5. Faculdade de Medicina de Botucatu, Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Botucatu, SP, Brazil

    Mariângela Marques

  6. LADR GmbH MVZ Dr, Kramer & Kollegen, Lauenburger Straße 67, 21502, Geesthacht, Germany

    Ralf Bialek

  7. Departamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Campus Diadema, São Paulo, SP, Brazil

    Wagner Luiz Batista

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Contributions

GR: conceptualization, funding acquisition, methodology, formal analysis, writing-original draft, writing-review and editing, supervision; EBC: conceptualization, methodology, formal analysis, writing-original draft, writing-review and editing, supervision; ASN: methodology, writing-original draft, writing-review, and editing; RP: resources, writing-original draft, writing-review and editing; MM: resources, writing-original draft, writing-review, and editing; RB: conceptualization, methodology, writing-original draft, writing-review, and editing; AMR: methodology, formal analysis, writing-original draft, writing-review, and editing; MF: conceptualization; WLB: conceptualization, methodology, formal analysis, writing-original draft, writing-review and editing, supervision. All the authors approved the final version of the manuscript, including the authorship list.

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Correspondence to Giannina Ricci.

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Ricci, G., Campanini, E.B., Nishikaku, A.S. et al. PbGP43 Genotyping Using Paraffin-Embedded Biopsies of Human Paracoccidioidomycosis Reveals a Genetically Distinct Lineage in the Paracoccidioides brasiliensis Complex. Mycopathologia 187, 157–168 (2022). https://doi.org/10.1007/s11046-021-00608-3

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