Abstract Background <it>Paracoccidioides brasiliensis </it>is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi. Results A cDNA (<it>Pb</it>sp) encoding a secreted serine protease (<it>Pb</it>SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant <it>Pb</it>SP was produced in <it>Escherichia coli</it>, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the <it>P. brasiliensis </it>proteome. <it>In vitro </it>deglycosylation assays with endoglycosidase H demonstrated that <it>Pb</it>SP is a <it>N</it>-glycosylated molecule. The <it>Pb</it>sp transcript and the protein were induced during nitrogen starvation. The <it>Pb</it>sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of <it>Pb</it>SP with <it>P. brasiliensis </it>proteins were evaluated by two-hybrid assay in the yeast <it>Saccharomyces cerevisiae</it>. <it>Pb</it>SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2. Conclusions A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. <it>Pb</it>SP interactions with other <it>P. brasiliensis </it>proteins were reported. Proteins interacting with <it>Pb</it>SP are related to folding process, protein trafficking and cytoskeleton reorganization.</p

Bailão, Alexandre M

Borges, Clayton L

Parente, Juliana A

Pereira, Maristela

Salem-Izacc, Sílvia M

Santana, Jaime M

Soares, Célia MA

English

PubMed

Abstract Background Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi. Results A cDNA (Pbsp) encoding a secreted serine protease (PbSP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant PbSP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that PbSP is a N-glycosylated molecule. The Pbsp transcript and the protein were induced during nitrogen starvation. The Pbsp transcript was also induced in yeast cells infecting murine macrophages. Interactions of PbSP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. PbSP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2. Conclusions A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. PbSP interactions with other P. brasiliensis proteins were reported. Proteins interacting with PbSP are related to folding process, protein trafficking and cytoskeleton reorganization.</p

Soares Célia MA

Bailão Alexandre M

Borges Clayton L

Pereira Maristela

Santana Jaime M

Salem-Izacc Sílvia M

Parente Juliana A

Directory of Open Access Journals

BMC Microbiology

A secreted serine protease of <it>Paracoccidioides brasiliensis </it>and its interactions with fungal proteins

Juliana A Parente

Sílvia M Salem-Izacc

Jaime M Santana

Maristela Pereira

Clayton L Borges

Alexandre M Bailão

Célia MA Soares

Springer - Publisher Connector

A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins

Abstract
 
 Background
 
 Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi.
 
 
 Results
 A cDNA (Pb sp) encoding a secreted serine protease (Pb SP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant Pb SP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that Pb SP is a N-glycosylated molecule. The Pb sp transcript and the protein were induced during nitrogen starvation. The Pb sp transcript was also induced in yeast cells infecting murine macrophages. Interactions of Pb SP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. Pb SP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2.
 
 
 Conclusions
 A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. Pb SP interactions with other P. brasiliensis proteins were reported. Proteins interacting with Pb SP are related to folding process, protein trafficking and cytoskeleton reorganization.
 </jats:sec

Crossref

Background Paracoccidioides brasiliensis is a thermodimorphic fungus, the causative agent of paracoccidioidomycosis (PCM). Serine proteases are widely distributed and this class of peptidase has been related to pathogenesis and nitrogen starvation in pathogenic fungi. 
Results A cDNA (Pbsp) encoding a secreted serine protease (PbSP), was isolated from a cDNA library constructed with RNAs of fungal yeast cells recovered from liver of infected mice. Recombinant PbSP was produced in Escherichia coli, and used to develop polyclonal antibodies that were able to detect a 66 kDa protein in the P. brasiliensis proteome. In vitro deglycosylation assays with endoglycosidase H demonstrated that PbSP is a N-glycosylated molecule. The Pbsp transcript and the protein were induced during nitrogen starvation. The Pbsp transcript was also induced in yeast cells infecting murine macrophages. Interactions of PbSP with P. brasiliensis proteins were evaluated by two-hybrid assay in the yeast Saccharomyces cerevisiae. PbSP interacts with a peptidyl prolyl cis-trans isomerase, calnexin, HSP70 and a cell wall protein PWP2. 
Conclusions A secreted subtilisin induced during nitrogen starvation was characterized indicating the possible role of this protein in the nitrogen acquisition. PbSP interactions with other P. brasiliensis proteins were reported. Proteins interacting with PbSP are related to folding process, protein trafficking and cytoskeleton reorganization

Parente, Juliana Alves

Salem-Izacc, Sílvia Maria

Santana, Jaime Martins de

Borges, Clayton Luiz

Bailão, Alexandre Melo

Soares, Célia Maria de Almeida

Repositório Institucional da Universidade de Brasília

AJ: Evolutionary families of peptidases.

Caddick MX: Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA. Mol Microbiol

characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis. Microbiol Res

Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature

CMA: Proteomic identification, nucleotide sequence, heterologous expression and immunological reactivity of the triosephosphate isomerase of Paracoccidioides brasiliensis. Microbes Infect

DB: In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control. Methods

Degelmann A: Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorpha. FEMS Yeast Res

Essential roles of 70 kDa heat inducible proteins. Bioessays

et al: Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction.

Fink GR: Phagocytosis by neutrophils induces an amino acid deprivation response in Saccharomyces cerevisiae and Candida albicans. Proc Natl Acad Sci USA

GD: Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wallassociated and expressed during infection of macrophages.

Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium.

Helenius A: Quality control in the secretory pathway: the role of calreticulin, calnexin and BiP in the retention of glycoproteins with C-terminal truncations. Mol Biol Cell

JD: PWP2, a member of the WD-repeat family of proteins, is an essential Saccharomyces cerevisiae gene involved in cell separation. Mol Gen Genet

JM: Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli. Mol Microbiol

Marzluf GA: Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit3 gene, which encodes nitrate reductase in Neurospora crassa.

Meira DA: Host-parasite relationship in paracoccidioidomycosis. Curr Top Med Mycol

MF: Highthroughput real-time quantitative reverse transcription PCR. In Current Protocols in Molecular Biology. Edited by: Ausubel

Monod M: Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus infection.

Nosanchuk JD: Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol

RA: Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea. Fungal Genet Biol

Russel DW: Molecular Cloning. A Laboratory Manual.

Serum stimulates growth of and proteinase secretion by Aspergillus fumigatus. Infect Immun

Soares CMA: Characterization of a secreted aspartyl protease of the fungal pathogen Paracoccidioides brasiliensis. Med Mycol

Soares CMA: Detection of a homotetrameric structure and proteinprotein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insights. FEMS Yeast Res

Soares CMA: Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: representational difference analysis identifies candidate genes associated with fungal pathogenesis. Microbes Infect

Soares CMA: The transcriptional profile of Paracoccidioides brasiliensis yeast cells is influenced by human plasma. FEMS Immunol Med Microbiol

Soares CMA: Transcriptome overview of Paracoccidioides brasiliensis proteases. Genet Mol Res

Soares CMA: Twodimensional electrophoresis and characterization of antigens from Paracoccidioides brasiliensis. Microbes Infect

Soares RM: Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization. J Oral Pathol Med

Travassos LR: Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis.

Travassos LR: Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane. Med Mycol

Zhang KQ: Regulation of subtilisin-like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea. Environ Microbiol

http://repositorio.unb.br/bitstream/10482/11978/3/ARTIGO_SecretedSerineProtease.pdf

A secreted serine protease of Paracoccidioides brasiliensis and its interactions with fungal proteins

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

Springer - Publisher Connector

Crossref

Repositório Institucional da Universidade de Brasília