Identification of a Metallopeptidase with TOP-Like Activity in Paracoccidioides Brasiliensis, with Increased Expression in A
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Medical Mycology January 2012, 50, 81–90 Identifi cation of a metallopeptidase with TOP-like activity in Paracoccidioides brasiliensis, with increased expression in a virulent strain ELLEN T. GRAVI * , THAYSA PASCHOALIN * , BIANCA R. DIAS * , DAYSON F. MOREIRA ‡ , JOS É E. BELIZARIO ‡ , VITOR OLIVEIRA † , ADRIANA K. CARMONA † , MARIA A. JULIANO † , LUIZ R. TRAVASSOS * & ELAINE G. RODRIGUES * * Unidade de Oncologia Experimental (UNONEX), Departamento de Microbiologia, Imunologia e Parasitologia, and Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 † Departamento de Biof í sica, Universidade Federal de S ã o Paulo-Escola Paulista de Medicina (UNIFESP-EPM), Brazil, and ‡ Departamento de Farmacologia, Universidade de S ã o Paulo (USP), Brazil Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis , is a systemic mycosis with severe acute and chronic forms. The pathology of PCM is not completely understood, and the role of proteases in the infection is not clearly defi ned. In this report, we describe a metallopeptidase activity in P. brasiliensis total and cytosolic protein extracts similar to that of mammalian thimet oligopeptidase (TOP). The analogous enzyme was suggested by analysis of P. brasiliensis genome data- bank and by hydrolytic activity of the FRET peptide Abz-GFSPFRQ-EDDnp which was completely inhibited by o -phenanthrolin and signifi cantly inhibited by the TOP inhibitor, JA-2. This activity was also partially inhibited by IgG purifi ed from patients with PCM, but not from normal individuals. As shown by high-performance liquid chromatography (HPLC), the hydrolysis of bradykinin had the same pattern as that of mammalian TOP, and anti-mammalian TOP antibodies signifi cantly inhibited fungal cytosolic peptidase activity. Moreover, anti-mammalian TOP antibodies recognized a component of 80 kDa on fungal cytosol. A P. brasiliensis virulent isolate showed higher gene expression and TOP-like peptidase activity than a non-virulent strain. The release of enzyme following fungal lysis would be consistent with host antibody production and may have a role in the pathogenesis, infl ammation and further development of the mycosis. Keywords Paracoccidioides brasiliensis , metallopeptidase , thimet oligopeptidase , bradykinin , FRET peptides Introduction the airbone propagules derived from the mycelial saprophytic stage of P. brasiliensis [2]. In the infected The dimorphic fungus Paracoccidioides brasiliensis is the host, the fungus undergoes transition to the yeast form, etiologic agent of paracoccidioidomycosis (PCM), the pre- an essential step for the establishment of the pulmonary dominant systemic mycosis in Central and South America infection [3]. [1]. Infection is probably acquired by inhalation of conidia, So far, a few proteases have been isolated from P. brasil- iensis and the role of these molecules in fungal biology and Received 31 October 2010; Received in fi nal revised form 18 March infection is still unclear. It has been suggested that fungal 2011; Accepted 20 May 2011 proteases may be involved in the pathogenesis of the dis- Correspondence: Elaine Guadelupe Rodrigues, Unidade de Oncologia ease despite the fact that the enzymatic activity of these Experimental, Departamento de Microbiologia, Imunologia e Parasito- molecules was not further investigated. However, it is logia, Universidade Federal de S ã o Paulo-Escola Paulista de Medicina (UNIFESP-EPM), Rua Botucatu, 862, 8 ° andar, S ã o Paulo, SP 04023- known that the 32-kDa hydrolase was implicated in the 062, Brazil. Tel: ϩ 55 11 5084 2991 ext 14; Fax: ϩ 55 11 5571 5877; adherence of P. brasiliensis to human epithelial cells [4], E-mail: [email protected] the malate synthase behaves as an anchorless adhesin [5] © 2012 ISHAM DOI: 10.3109/13693786.2011.590825 82 Gravi et al . and enolase was identifi ed as a fi bronectin-ligand protein Materials and methods [6]. Secreted serine and aspartyl proteases have unknown Phylogeny studies biological functions [7,8], and a serine thiol-proteinase may facilitate the invasion of host tissues by P. brasiliensis Sequence data from M3 families of metallopeptidases were by degrading components of the extracellular matrix obtained from NCBI database (http://www.ncbi.nlm.nih. (ECM) [9]. gov/genbank/) and aligned using MegaAlign (DNAStar, Recent studies based on the identifi cation of protease Lasergene). The sequences and accession numbers used for genes in the transcriptome of P. brasiliensis isolate 01 the phylogenetic analysis were: thimet oligopeptidase from showed the presence of numerous proteases, and 23% of P. brasiliensis isolate 01 (EEH41798.1) and isolate 03 them belonged to the family of metallopeptidases [10]. (EEH18209.1); saccharolysin from P. brasiliensis isolate Among the metallopeptidases, thimet oligopeptidase or 18 (EEH47123.1); saccharolysin from Sacharomices TOP (EC 3.4.24.15) is a thiol-dependent endooligopepti- cerevisiae (CAA54039.1); human thimet oligopeptidase Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 dase that belongs to the M3A subfamily. TOP hydrolyzes (AAH13878.1); human neurolysin (NP_065777.1); Escher- several bioactive peptides including enkephalin, luliberin ichia coli oligopeptidase A (NP_417955); mitochondrial and bradykinin [11 – 13]. The mitochondrial intermediate intermediate peptidase from P. brasiliensis isolate 01 peptidase (MIP – EC 3.4.24.59) is a metallopeptidase (EEH38189.1), isolate 03 (EEH19367.1), isolate 18 homologue to TOP [14] and is also capable of cleaving (EEH47637.1) and human (NP_005923.2). A cladogram bradykinin [15]. In Saccharomyces cerevisiae, a soluble was generated with the alignment results. metallopeptidase called oligopeptidase yscD has been described, showing 34.8% identity and 55% similarity with P. brasiliensis isolates and growth conditions the sequence of rat TOP metallopeptidase [16,17]. An intracellular metallopeptidase of 82 kDa named MepB, Highly virulent P. brasiliensis Pb18 isolate (Pb18V) was also related to TOP, has been described in Aspergillus recovered from lungs of BALB/c male mice (6 – 8 weeks) fumigatus [18]. This peptidase is able to cleave collagen which had been inoculated intravenously (i.v.), and another type I, and shows 45% and 41% identity with mammalian isolate of lower virulence had been cultivated in vitro for TOP and neurolysin, respectively. The identity with bacte- several years (Pb18NV). Yeast cells were maintained and rial oligopeptidases is about 30% [19]. cultivated in solid modifi ed YPD medium (0.5% yeast Bradykinin (BK) is a bioactive peptide released from extract, 0.5% casein peptone, 1.5% glucose, pH 6.5) at the kininogen precursor by the action of kallikreins that is 37 ° C. Fungal cells were recovered from infected organs at involved in infl ammation, increased vascular permeability, 36 ° C as described previously [22]. stimulation of the endothelial isoform of nitric oxide (NO) synthase, vasodilation, and may also have a role in the Preparation of fungal protein extracts control of Trypanosoma cruzi infection [20]. The parasite secretes cruzipain, a cysteine protease able to release kinins Total protein extract was obtained from approximately from kininogen accumulated at the sites of infection after 0.1 ml of wet yeast pellet by cell disruption with glass beads recruitment of infl ammatory cells [20]. Bradykinin acts as (425-600 μ M, Sigma-Aldrich, St Louis, MO) in Tris-buffered a short-lived ‘ danger ’ signal and stimulates dendritic cells solution (TBS, 10mM Tris-HCl, 150mM NaCl, pH 7.4), and via the bradykinin B2 receptor (B2R) to produce interleu- centrifugation at 12,000 g for 5 min. Alternatively this mate- kin-12 (IL-12), demonstrating that endogenous kinins gen- rial was ultracentrifuged at 100,000 g for 2 h. The pellet was erated in the injured tissues may induce a T helper 1 resuspended in 50 mM Tris-HCl, pH 7.4 ( membrane protein (Th1)-type immune response [21]. As the development of extract ) and the supernatant was called the cytosolic protein a Th1-type immune response is critical for the ability of extract. Protein concentration of samples was determined by the host to eliminate P. brasiliensis [22,23], the presence a modifi ed Bradford micromethod [24] using bovine serum of a BK-degrading protease may negatively modulate the albumin as standard. In all assays, 10 to 15 μ g of cytosolic protective response induced in vivo thus facilitating fungal protein extract or total protein extract were used for enzymatic development. activity determination. In the present work, we characterized the activity of a metallopeptidase similar to mammalian TOP from FRET peptides P. brasiliensis , showing that the peptidase has a higher expression and activity in a virulent strain than in a non- Peptides were synthesized at the Department of Biophysics, virulent strain. In addition, we demonstrated the hydrolysis UNIFESP. FRET (Fluorescence resonance energy transfer) of BK by the TOP-like peptidase from P. brasiliensis which peptides having attached to the N- and C-terminal groups, may be involved in the disease development in vivo . respectively, ortho -aminobenzoic acid (Abz) as fl uorescent © 2012 ISHAM, Medical Mycology, 50, 81–90 TOP-like metallopeptidase of P. brasiliensis 83 group and ethylenediamino-2,4-dinitrophenyl (EDDnp) as Purifi ed IgG (100 μ g), from the serum of individual quencher were synthesized by the solid-phase synthesis patients with different immunodiffusion titers (1:2, 1:4, 1:16 method and analyzed for purity as described elsewhere [25]. and 1:128) and from the pool of healthy controls were added to Pb18V cytosolic protein extract and incubated for 10 min- utes at 37 ° C. After incubation, the substrate Abz-GFSPFRQ- Kinetic assays EDDnp was added and the fl uorescence was monitored as Hydrolysis of FRET peptides (approx. 20 μ M) at 37 o C in described above. The percent inhibition was calculated 50 mM Tris-HCl buffer, pH 7.4, was followed by measur- considering as 100% the reaction of the Pb18V cytosolic λ ϭ λ ϭ ing the fl uorescence at em. 420 nm and ex. 320 nm protein extract with substrate Abz-GFSPFRQ-EDDnp.