Expression of Hsp60 and its cell location in Paracoccidioides brasiliensis

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.

Paracoccidioides brasiliensis; Heat-shock proteins; Hsp60; Subcellular localization

Authorship

Fabrício Freitas Fernandes

Universidade Paulista, Instituto de Ciências da Saúde, Ribeirão Preto, São Paulo, BrazilUniversidade PaulistaBrazilRibeirão Preto, São Paulo, BrazilUniversidade Paulista, Instituto de Ciências da Saúde, Ribeirão Preto, São Paulo, Brazil

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, São Paulo, Brazil

Nayla S. Pitangui

Gabriela Peron

Universidade Estadual de Campinas, Instituto de Biologia, Campinas, São Paulo, BrazilUniversidade Estadual de CampinasBrazilCampinas, São Paulo, BrazilUniversidade Estadual de Campinas, Instituto de Biologia, Campinas, São Paulo, Brazil

Clarissa X. R. Valim

Vívian M. Mazucato

Aline F. Oliveira

Thiago A. da Silva

Maria-Cristina Roque-Barreira

Ademilson Panunto-Castelo

Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, São Paulo, Brazil

http://orcid.org/0000-0003-0148-0133

Correspondence to: Ademilson Panunto-Castelo Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Av. Bandeirantes, 3900, CEP 14040-901 Ribeirão Preto, SP, Brazil E-mail:apcastelo@usp.br

CONFLICT OF INTERESTS

On behalf of all of the authors, the corresponding author states that there is no conflict of interests.

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (3)

Thumbnail

Figure 1
Differential expression of the HSP60 mRNA in the different morphologies and transition phases of P. brasiliensis. Optical microscopy of mycelia (A), mycelia-to-yeast (B), yeast-to-mycelia (C), yeast (D) of P. brasiliensis. The bars correspond to 10 μm. (E) Differential expression of HSP60 mRNA in the different morphologies and transition phases performed by qPCR. The abbreviation M refers to mycelia, M→Y, mycelia-to-yeast, Y, yeast, and Y→M, yeast-to-mycelia. Data are expressed as the mean ± standard deviation of five samples per group. *P < 0.05 compared to the mycelia and yeast-to-mycelia groups.

Thumbnail

Figure 2
Expression of rPbHsp60 in P. brasiliensis and analysis of the reactivity with anti-rPbHsp60 polyclonal serum: A) pET28a-HSP60-transformed E. coli were cultured in LB broth in the absence (NI) or presence (IPTG) of the inductor of the recombinant protein for 6 h at 37 oC under constant agitation. The recombinant protein was purified (P) on a HisTrap column. The preparations were analyzed in an SDS-PAGE and the proteins visualized by staining with Coomassie brilliant blue G250 (USB Corporation, Cleveland, OH, USA); B) Whole soluble antigens (PbAg) and recombinant protein (rPBHsp60) were separated by 12% SDS-PAGE, blotted blotted onto a PVDF membrane and probed with an anti-rPbHsp60 polyclonal antibody. The antigen-antibody reactions were revealed with the ECL reagent.

Thumbnail

Figure 3
Subcellular location of Hsp60 at P. brasiliensis by immunoelectron microscopy. (A-C) Yeast cells and (D-F) mycelium of P. brasiliensis were cultured in DMEM at 37 oC and 25 oC, respectively, for 7 days. Samples were fixed and incubated with (A and D) pre-immune serum or (B, C, E and F) anti-rPbHsp60 polyclonal antibody. The arrows show the PbHsp60 within the cell wall (CW) of P. brasiliensis. Scale bar = 2 μm.

Figure 1 Differential expression of the HSP60 mRNA in the different morphologies and transition phases of P. brasiliensis. Optical microscopy of mycelia (A), mycelia-to-yeast (B), yeast-to-mycelia (C), yeast (D) of P. brasiliensis. The bars correspond to 10 μm. (E) Differential expression of HSP60 mRNA in the different morphologies and transition phases performed by qPCR. The abbreviation M refers to mycelia, M→Y, mycelia-to-yeast, Y, yeast, and Y→M, yeast-to-mycelia. Data are expressed as the mean ± standard deviation of five samples per group. *P < 0.05 compared to the mycelia and yeast-to-mycelia groups.

Figure 2 Expression of rPbHsp60 in P. brasiliensis and analysis of the reactivity with anti-rPbHsp60 polyclonal serum: A) pET28a-HSP60-transformed E. coli were cultured in LB broth in the absence (NI) or presence (IPTG) of the inductor of the recombinant protein for 6 h at 37 oC under constant agitation. The recombinant protein was purified (P) on a HisTrap column. The preparations were analyzed in an SDS-PAGE and the proteins visualized by staining with Coomassie brilliant blue G250 (USB Corporation, Cleveland, OH, USA); B) Whole soluble antigens (PbAg) and recombinant protein (rPBHsp60) were separated by 12% SDS-PAGE, blotted blotted onto a PVDF membrane and probed with an anti-rPbHsp60 polyclonal antibody. The antigen-antibody reactions were revealed with the ECL reagent.

Figure 3 Subcellular location of Hsp60 at P. brasiliensis by immunoelectron microscopy. (A-C) Yeast cells and (D-F) mycelium of P. brasiliensis were cultured in DMEM at 37 oC and 25 oC, respectively, for 7 days. Samples were fixed and incubated with (A and D) pre-immune serum or (B, C, E and F) anti-rPbHsp60 polyclonal antibody. The arrows show the PbHsp60 within the cell wall (CW) of P. brasiliensis. Scale bar = 2 μm.

How to cite

copy

Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 - São Paulo - SP - Brazil, Tel. +55 11 3061-7005 - São Paulo - SP - Brazil
E-mail: revimtsp@usp.br

Acompanhe os números deste periódico no seu leitor de RSS

Versão para download de PDF

PDF

Inglês

Versões e tradução automática

Versão original do texto

English

Tradução automática

Google Translator
Microsoft Translator

Como citar

RIS

BIBTEX

Outros formatos de citação e exportação:

SciELO - Scientific Electronic Library Online
Rua Dr. Diogo de Faria, 1087 – 9º andar – Vila Clementino 04037-003 São Paulo/SP - Brasil
E-mail: scielo@scielo.org

Leia a Declaração de Acesso Aberto

Métricas

Expression of Hsp60 and its cell location in Paracoccidioides brasiliensis

PlumX

Mensagem

[1] Correspondence to: Ademilson Panunto-Castelo Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Av. Bandeirantes, 3900, CEP 14040-901 Ribeirão Preto, SP, Brazil E-mail:apcastelo@usp.br