Identification of a Metallopeptidase with TOP-Like Activity in Paracoccidioides Brasiliensis, with Increased Expression in A

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‡ , JOSÉ 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 Biofí sica, Universidade Federal de Sã o Paulo-Escola Paulista de Medicina (UNIFESP-EPM), Brazil, and ‡ Departamento de Farmacologia, Universidade de Sã 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 databank 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. brasiliensis 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, Sã 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 μg), 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. in a Hitachi F-2000 spectrofl uorometer, as previously described [26]. The inhibitors PMSF (phenylmethylsulfo- Infection of BALB/c mice with P. brasiliensis isolates nyl fl uoride), E-64 [ trans -epoxysuccinyl-L-leucylamido- Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 (4-guanido)butane], o -phenanthrolin, Z-Pro-Prolinal, Male BALB/c mice (6 – 8 weeks old) were infected intra- ϫ 6 Pro-Ile and captopril {(2S )-1-[(2S )-2-methyl-3-sulfanyl- venously (i.v.) by the tail vein with 1 10 viable Pb18V propanoyl] pyrrolidine-2-carboxylic acid}, all purchased and Pb18NV yeast cells (verifi ed using Trypan blue exclu- μ from Sigma-Aldrich, were added to determine the relative sion) in 100 l of phosphate-buffered saline (PBS). Col- contribution of various proteases in the cleavage of ony-forming units (CFU) were determined after 7 days in substrates. N -[1-(R, S)-carboxy-3-phenylpropyl]-Ala-Aib- the lungs, homogenized and cultivated in supplemented Tyr-p -aminobenzoate (JA-2), an inhibitor of human TOP Brain Heart Infusion Medium (BHIM) agar as described Ϯ [27] was obtained from Ian Smith, Baker Heart Research [22]. The results are shown as the average value Institute, Australia. The results were recorded as the per- standard deviation of fi ve animals/group. centage of residual activity relative to control reactions in the absence of the inhibitor, and the standard deviations Chemiluminescent (CL)-immunoblotting were not higher than 5% in all samples analyzed. Recombinant mammalian TOP [26] and P. brasiliensis total cytosolic extract were separated in 10% SDS-PAGE Determination of cleaved peptide bonds and electro-transferred onto nitrocellulose membranes (0.2 μ m, Amersham Bioscience, England). Membranes were The hydrolysis of BK (RPPGFSPFR) (50 μ M) and its incubated for 1 h in PBS and 5% dry skim milk. The anti- derived FRET peptide Abz-GFSPFRQ-EDDnp (20 μ M) by mammalian TOP (1:1000; Proteimax, Sã o Paulo, Brazil) Pb18V cytosolic protein extract was carried out as described was used as the primary antibody, and the secondary anti- [26]. The scissile bonds of hydrolyzed peptides were iden- body was horse-radish peroxidase-conjugated anti-rabbit tifi ed by the isolation of the fragments by high-performance antibody in PBS and 1% dry skim milk. The blot was visu- liquid chromatography (HPLC) followed by determination alized using a chemiluminescent substrate (Millipore). of their molecular mass in a mass spectrometer LCMS- 2010 equipped with an electronspray ionization (ESI)- probe (Shimadzu, Tokyo, Japan). P. brasiliensis RNA extraction RNA from isolates Pb18V and Pb18NV was obtained after Purifi cation of IgG from sera of patients with 5 days growth on YPD agar medium. Pb18V was grown in paraccocidioidomycosis and peptidase inhibition assay vitro and subcultured 3– 6 times. Wet yeast pellet (0.1 ml) was mechanically disrupted by vortexing with 0.1 ml of Sera from patients with paracoccidioidomycosis were glass beads for 10 min in the presence of 0.7 ml of TRIzol kindly provided by Arnaldo L. Colombo, Infectious Dis- (Invitrogen, Carlsbad, CA). After incubation for 10 min at eases Department, Federal University of Sã o Paulo, Brazil. room temperature, cellular debris was removed by centrifu- The titers of specifi c P. brasiliensis immunoglobulins (Ig) gation at 14,000 g for 10 min at 4° C. The supernatant was were determined by immunodiffusion test [28]. A pool of then processed for RNA purifi cation following the manu- fi ve sera from healthy volunteers, non-reactive with P. facturer’ s instructions. RNA preparation was quantifi ed by brasiliensis antigens, was used as control. Human IgGs spectrometry and analyzed for integrity in Agarose gel. were purifi ed using HiTrap Protein G columns (GE Health- care, Uppsala, Sweden) following the manufacturer’ s Quantitative real time RT-PCR (q-PCR) instructions. The purity of the preparation was analyzed in sodium dodecyl sulfate polyacrylamide gel electrophoresis Syber Green detection system (Invitrogen, Carlsbad, (SDS-PAGE), and protein concentration measured as CA, USA) was used in quantitative real time RT-PCR exper- described [24]. iments, following the manufacturer ’ s instructions. The cDNA

© 2012 ISHAM, Medical Mycology, 50, 81–90 84 Gravi et al . synthesis from total RNA (2 μg) treated with DNase (Ambion, Results Austin, TX, USA) was obtained using the SuperScript III Phylogenetic analysis First strand synthesis (Invitrogen). After cDNA synthesis, the reaction was diluted 5-fold with ultrapure water. Primers To estimate the evolutionary distance between bacterial/ were designed based on sequences of the respective homolo- fungal TOP-like and mammalian TOP peptidases, a clado- gous genes deposited on P. brasiliensis Pb01, Pb03 and Pb18 gram was constructed using the nucleotide sequence align- genome bank (http://www.broadinstitute.org/annotation/ ment performed by MegAlign software. The phylogenetic genome/paracoccidioides_brasiliensis/MultiHome.html) and tree is shown in Fig. 1. Although all peptidases included in were used at 250 nM: 5´ -GAGCAAATTTCGGCAACA-3 ´ the alignment belong to the same family (M3), the analysis and 5´-GGAAAACAGCCGTAGTAGGCTTAA-3 ´ for TOP, clearly shows the division of two distinct groups, derived 5´ -TCAACTACGGCGCCACCTA-3´ and 5´ -TCTCAGCC from the same common ancestor. The fi rst group comprises

ATAGCTTACTTGCT-3´ for MIP, 5´ -TATACCGCCACCC the MIPs of the three isolates of P. brasiliensis and the Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 AGAAGAC-3´ and 5´ -TGAGAGCTGGGATGACCTTT-3 ´ second group the other evaluated metallopeptidases. for GAPDH. The gene annotated as Saccharolysin in the Pb18 The TOP-like metallopeptidase from P. brasiliensis genome has more than 90% identity with the TOP-like metal- have higher identity with saccharolysin from S. cerevisiae lopeptidase gene from Pb01 and Pb03 genomes, and for this than with mammalian oligopeptidases. All these fungal reason it was used for designing primers for TOP. The regions predicted protein sequences exhibit key TOP features such selected for primer annealing were 100% identical for all three as the presence of the highly conserved catalytic motif isolates. P. brasiliensis glyceraldehyde 3-phosphate dehydro- HEXXH, in which the histidine residues coordinate the genase (GAPDH) and mitochondrial intermediate peptidase zinc ion, and a downstream glutamate residue serves as the (MIP) genes were used to normalize the reaction. Cycling was third zinc ligand (data not shown). The glutamate residue carried out in a Stratagene Mx3005P System (Stratagene, CA, of the HEXXH motif is believed to play an important USA), starting with one cycle of 95 ° C (10 min), followed by role in the reaction mechanism, polarizing and orienting a 40 or 50 cycles at 95° C (30 s) and 60° C (30 s). The dissocia- zinc-coordinating water molecule [31]. tion curve (melting curve analysis) was determined with an Interestingly, a gene annotated as Saccharolysin in the additional cycle of 95 ° C (1min), ranging from 55 ° C to 95 ° C, Pb18 genome, has a high identity with the TOP-like metal- with temperature increasing by steps of 0.5 ° C. Negative con- lopeptidase gene from Pb03 and Pb01 genomes and pres- trols did not contain DNA or RNA. Reactions were run in ents the key TOP features described above (Fig. 1 and data duplicates with three biological samples for each group (viru- not shown). lent and non-virulent). The cycle threshold (Ct) values were automatically determined for each gene using the MxPro- Metallopeptidase activity in the total protein extract Mx3005P v3.0 (Stratagene). The resulting data were con- and cytosolic protein extract from P. brasiliensis: verted into correct input fi les, according to the requirements similarity with TOP of the software, and analyzed with the VBA applet geNorm [29]. To ensure correct normalization of the expression levels To characterize the metallopeptidase activity of P. brasilien- for the target genes, the stability of the two housekeeping sis total protein extract and cytosolic protein extract, FRET (HKG) genes, GAPDH and MIP, was statistically determined peptides derived from neurotensin (NT) and bradykinin using the VBA applet geNorm [29]. The REST 2008 v2.0.7 (BK), previously used to determine the specifi city of recom- software [30] was used to compare the expression levels of binant mammalian TOP and neurolysin oligopeptidases the metallopeptidase genes in both fungal groups (virulent and [26,32 – 34], were used. Total protein extract and cytosolic non-virulent) using the two HKGs previously analyzed by protein extract from Pb18 virulent strain (Pb18V) were able geNorm . The expression ratio of the investigated transcripts to cleave all the BK-derived substrates tested, Abz-GFSP- was tested for signifi cance by a Pair Wise Fixed Reallocation FRQ-EDDnp, Abz-GFSIFRQ-EDDnp and Abz-GFPPFRQ- Randomization Test and plotted using standard error (SE) esti- EDDnp, as previously demonstrated for mammalian TOP mation via a complex Taylor algorithm [30]. Samples with a (Fig. 2A, Supplementary Table 1 – available online only). probability value of Ͻ 0.05 were considered signifi cantly dif- Both extracts cleaved preferentially Abz-GFSPFRQ- ferent. This assay was repeated twice, with similar results. EDDnp, which is a very susceptible substrate for mammalian TOP. In contrast, poor hydrolysis (below 10%) of Abz-NKPRRPQ-EDDnp, a peptide derived from NT which Statistical analysis is very susceptible to neurolysin, was observed. In addition, The results are expressed as mean values Ϯ SD. We used the neprilysin selective substrate Abz-rRL-EDDnp [35] was two-sided Student ’ s t test for statistical analysis of data and not hydrolysed by P. brasiliensis total protein extract, P Ͻ 0.05 for signifi cance. neither by mammalian TOP (Supplementary Table 1).

Fig. 1 Phylogenetic tree of M3 metallopeptidases. The sequences and accession numbers used for the phylogenetic analysis are described in the Material and Methods section. Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021

Therefore, the hydrolysis of the FRET peptides by the No hydrolytic activity of FRET peptides was observed Pb18V cytosolic protein extract showed greater similarity using fungal culture supernatant, including a chemically with that of mammalian TOP than those of mammalian defi ned medium enriched with fetal calf serum. The fungal neurolysin and bacterial OpdA [33,36 and Supplementary membrane protein extract also did not show hydrolytic Fig. 1]. activity with the same peptides (data not shown). These

Fig. 2 Presence of a TOP-like activity in Pb18V total protein extract and cytosolic protein extract. (A) Pb18V total protein extract and cytosolic protein λ ϭ λ ϭ extract were incubated with different FRET peptides. Hydrolysis was followed by measuring the fl uorescence at em 420 nm and ex. 320 nm. (B) BK was incubated with Pb18V cytosolic protein extract, reaction products were separated by HPLC and identifi ed by mass spectrometry. (C) Abz- λ ϭ GFSPFRQ-EDDnp was incubated with Pb18V cytosolic protein extract. Chromatogram developed by fl uorescence detection at em 420 nm and λ ϭ ex 320 nm. Chromatograms represent the untreated substrates (upper) and Pb18V cytosolic protein extract reaction products (lower). This experiment was repeated twice.

© 2012 ISHAM, Medical Mycology, 50, 81–90 86 Gravi et al . results suggest that Pb18 virulent strain has a major unse- 80 kDa present in the fungal cytosolic protein extract, creted TOP-like metallopeptidase activity in the cytosolic similar to that with the recombinant mammalian enzyme protein extract. [26] was observed (Fig. 3A). No cross-reactivity was To further confi rm the presence of a TOP-like activity observed with other fungal proteins. This antibody par- on P. brasiliensis, the Pb18V cytosolic protein extract was tially inhibited the fungal proteolytic activity (Fig. 3B). incubated with BK and a BK-derived FRET peptide, and the resulting cleaved fragments were analyzed. As observed previously with mammalian TOP and MIP [15,26,32], BK was cleaved by Pb18V cytosolic protein extract at the Phe-

Ser bond generating a peptide with fi ve residues (BK1-5 ) (Fig. 2B). Using a BK-derived FRET peptide, Abz-GFSP-

FRQ-EDDnp, Pb18V cytosolic protein extract showed an Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 activity similar to mammalian TOP [26,32], cleaving the substrate at the Pro-Phe bond (Fig. 2C). Protease inhibitors were assayed for their effects on the hydrolysis of Abz-GFSPFRQ-EDDnp by P. brasiliensis cytosolic protein extract (Table 1). As expected, the specifi c metallopeptidase inhibitor, o -phenanthrolin was highly effective inhibiting the hydrolysis of the FRET peptide by Pb18V cytosolic protein extract. JA-2, initially described as a specifi c TOP inhibitor [27], showed more than 90% inhibition of the cytosolic activity. These results confi rm that the metallopeptidase activity found in the cytosolic protein extract of P. brasiliensis is primarily TOP-like. The residual activity in the fungal extracts using JA-2 inhibitor (9%) may indicate the presence of another metallopeptidase able to cleave the same fl uorogenic substrate, for instance a MIP-like peptidase [15]. PMSF, E-64, Z-Pro-Prolinal, and captopril showed little inhibitory effect. The activity of P. brasiliensis extract was inhibited in 55% by Pro-Ile.

Detection of P. brasiliensis TOP-like metallopeptidase by immunoblotting and inhibition of peptidase activity by immunoglobulins A strong reactivity of a commercial anti-mammalian TOP polyclonal antibody with a component of approximately

Table 1 Effect of protease inhibitors on the hydrolysis of Abz- GFSPFRQ-EDDnp by Pb18V total protein extract Inhibitors Hydrolysis (%) *

Control (no inhibitor) 100 Fig. 3 Anti-mammalian TOP antibodies and the IgG fraction from sera o -phenanthrolin (4 mM) 0 of paracoccidiodomycosis patients partially inhibit the TOP-like activity JA-2 (3 μ M) 9 in Pb18V cytosolic protein extract. (A) Recombinant mammalian TOP Pro-Ile (1 mM) 45 and Pb18V cytosolic protein extract were analyzed by chemiluminescent PMSF (0.1 mM) 92 (CL)-immunoblotting with anti-mammalian TOP. Anti-mammalian E-64 (0.1 mM) 86 TOP antibody (B) or IgG purifi ed from individual patients with Captopril (20 μ M) 72 paracoccidioidomycosis (PCM IgG) with immunodiffusion titers of 1:2, Z-Pro-Prolinal (1 μ M) 92 1:4, 1:16 and 1:128 (C) were previously incubated with Pb18V cytosolic protein extract and Abz-GFSPFRQ-EDDnp peptide was added. Normal * 100% hydrolysis represents the cleavage of 20 μ M of Abz-GFSPFRQ- rabbit IgG or purifi ed IgG from a pool of healthy individuals were used EDDnp by Pb18V total protein extract during 5 min at 37o C in 50 mM as control. The percentage of hydrolysis inhibition was calculated based Tris-HCl, pH 7.4. on the reactivity without the antibody. *P Ͻ 0.05 compared to control.

P. brasiliensis cytosolic protein extract was preincubated however cytosolic protein extract from the virulent isolate with IgGs isolated from serum of paracoccidioidomycosis showed 2.3-fold higher activity compared to the non-viru- patients showing different titers in the immunodiffusion lent isolate (Fig. 4B). assay, from 1:2 to 1:128, the FRET peptide Abz-GFSP- The expression of the gene homologous to the mam- FRQ-EDDnp was added and the inhibition of the cytosolic malian TOP in Pb18V and Pb18NV was evaluated by real- proteolytic activity was evaluated. Lower immunodiffusion time PCR assay (qPCR). The sequences of TOP, MIP and titers indicate lower concentrations of anti-P. brasiliensis GAPDH genes deposited on P. brasiliensis Genome data- specifi c immunoglobulins in the sera. There was a direct bank for Pb01, Pb03 and Pb18 were used to design all correlation between the concentration of yeast-specifi c IgG primers. and peptide cleavage inhibition (Fig. 3C). Using IgGs iso- The two housekeeping genes (HKGs), MIP and GAPDH, lated from a pool of healthy individuals, no inhibition of tested in this study showed optimal overall reliability, con-

peptide cleavage was observed (Fig. 3C). fi rmed by geNorm software [29]. Analyzing the relative Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 expression data obtained by qPCR, the expression of P. brasiliensis strain 18 TOP-like gene was almost twice as Comparison of metallopeptidase expression and activity in high in the virulent isolate as in the non-virulent isolate, virulent and non-virulent isolates of P. brasiliensis using both control genes (Fig. 4C). The virulent strain (Pb18V) was isolated from organs of intravenously infected mice, and used with no more than Discussion fi ve in vitro subculturings, while the non-virulent strain (Pb18NV) has been cultivated in vitro for years in our Oligopeptidases of the M3 subfamily of metallopeptidases laboratory. have a role in many biological and pathological processes. BALB/c mice were intravenously infected with the Although more than one hundred fungal peptidase same number of viable yeast forms of Pb18V and Pb18NV sequences belonging to this family are deposited in the strains, and the number of colony forming units (CFU) MEROPS bank [36], very few fungal metallo- oligopepti- recovered from lungs of these animals 2 weeks after infec- dases have been isolated and characterized. The genomic tion is shown in Fig. 4A. A signifi cantly higher number of library of P. brasiliensis [http://www.broadinstitute.org/ CFUs was retrieved from animals infected with Pb18V annotation/genome/paracoccidioides_brasiliensis/Multi- (3 ϫ 104 CFU per gram of pulmonary tissue) than from Home.html] show six gene sequences identifi ed in the M3 animals infected with the non-virulent isolate (10 3 CFU per subfamily of metallopeptidases. These sequences were gram of tissue). aligned with known peptidases of this subfamily: human The cytosolic protein extracts from both isolates were TOP (thimet oligopeptidase), neurolysin and MIP (mito- able to hydrolyze the substrate Abz-GFSPFRQ-EDDnp, chondrial intermediate peptidase), OpdA (oligopeptidase

Fig. 4 Analysis of TOP-like metallopeptidase activity and gene expression in virulent and non-virulent isolates of Paracoccidioides brasiliensis . (A) CFU of the Pb18V and Pb18NV strains recovered from lungs of BALB/c mice, 15 days after i.v. infection. * P Ͻ 0.001. (B) Pb18V and Pb18NV cytosolic fractions were incubated with Abz-GFSPFRQ-EDDnp and substrate cleavage was determined. Results are expressed as means Ϯ SD. * P Ͻ 0.05. (C) Relative expression of TOP homologous gene using qPCR. The ratio of Pb18V/Pb18NV expression of TOP-like metallopeptidase gene, normalized by MIP (black bar, standard error 1.068– 3.161) or GAPDH (white bar, standard error 1.005– 3.297) both used as reference gene expression, is shown. The assays were performed in duplicate, with three biological samples for each group, and were repeated twice with similar results.

A) from E. coli and saccharolysin from S. cerevisae . The Moreover, we also examined the fragments originated from phylogenetic tree constructed using the alignment data the cleavage of the FRET peptide, Abz-GFSPFRQ-EDDnp, shows the division into two distinct groups with a common in which the addition of glutamine causes a shift of the ancestor. The TOP-like metallopeptidase from P. brasilien- scissile bond from Phe-Ser to Pro-Phe when this substrate sis isolates 01 and 03 and saccharolysin from isolate 18 are is cleaved by TOP [26,32]. The hydrolysis of the same homologous to human TOP, and all these sequences show substrate by recombinant MIP [15], as well as by recom- the key features of Thimet oligopeptidases. binant OpdA [33], is maintained at the Phe-Ser bond. The The fi nding of a gene similar to mammalian TOP in Pb18V cytosolic protein extract showed an activity similar P. brasiliensis suggests the presence of a related metallo- to that of mammalian TOP, however, the presence of a peptidase in this microorganism. The annotated name minor MIP-like activity cannot again be discarded, in the thimet should wait a better characterization of the enzyme, face of the much greater activity of the fungal TOP-like

particularly the thiol dependence for reactivity. Presently, metallopeptidase. Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 we evaluated the presence of a TOP-like activity in By immunoblotting, anti-mammalian TOP antibodies P. brasiliensis , initially by studying the cleavage of brady- recognized a component of 80 kDa in the fungal cytosolic kinin (BK) and neurotensin (NT)-derived FRET peptides, extract, with the same molecular mass of recombinant employed in mammalian TOP characterization [26,32– 34]. mammalian TOP. This is a polyclonal rabbit antibody that Our results showed that the major peptidase activity pres- recognizes mammalian TOP, but not mammalian neurolysin, ent in the total protein extract and cytosolic protein extract an oligopeptidase with 66% of identity with TOP. Interest- of Pb18 virulent strain is very similar to mammalian TOP, ingly, these polyclonal antibodies were able to partially with the absence of neprilysin- and neurolysin-like activi- inhibit the P. brasiliensis peptidase cleavage of the Abz- ties because FRET peptides preferentially cleaved by these GFSPFRQ-EDDnp substrate, in a dose-dependent manner. peptidases were not hydrolyzed. Sera from patients with PCM contain antibodies capable The hydrolytic activity of the Pb18V extracts on BK- of inhibiting the TOP-like peptidase activity of P. brasil- derived FRET peptide Abz-GFSPFRQ-EDDnp was com- iensis cytosol. A patient serum with a low titer of anti-P. pletely inhibited by o -phenanthroline, indicating the metal brasiliensis crude antigen on immunodiffusion assay (1:2) dependence of this activity. The substrate hydrolysis was was less inhibitory in contrast to another serum with higher 90% inhibited by JA-2, an inhibitor capable of inhibiting titer (1:128) in the same assay. This result indicates that TOP, neurolysin and OpdA [26,27,33]. On evaluating the the metallopeptidase is immunogenic in vivo probably after effect of other selective protease inhibitors, we concluded fungal lysis or immune processing of yeast cells. We were that serine proteases, cisteine proteases and angiotensin not able to identify the TOP-like peptidase activity in con- converting enzyme (ACE) are not involved in the hydroly- ditioned in vitro culture supernatant even after enrichment sis of the BK-derived FRET peptide. Although the total with fetal calf serum but this does not exclude in vivo protein extract hydrolytic activity was partially inhibited secretion of the enzyme. by Pro-Ile, a dipeptide described as a specifi c inhibitor of Finally, we compared the expression and activity of the neurolysin [37], the FRET peptides preferentially cleaved TOP-like metallopeptidase in P. brasiliensis virulent and by this enzyme [26,38] were not hydrolysed. non-virulent strains. The difference in virulence between Marcondes and collaborators [15] demonstrated that Pb18V and Pb18NV strains was confi rmed after in vivo human MIP, another peptidase that belongs to the M3 sub- inoculation, where mice infected with the virulent strain family, hydrolyzes the Abz-GFSPFRQ-EDDnp peptide, and showed in comparison with the non-virulent strain 3,000- this activity is inhibited by o -phenanthroline but it is not fold more fungal CFU recovered from the lungs. The cyto- inhibited by JA-2. As sequences with high identity to mam- sol from Pb18V and Pb18NV showed TOP-like peptidase malian MIP were found in the genome of P. brasiliensis activity 2.3 times higher in the virulent strain. Also, the strains 01, 03 and 18 (deposited in the genome databank), expression of the TOP-homologous gene was almost 2 and our results showed 90% inhibition of the hydrolytic times increased in Pb18 virulent strain, using both selected activity of Pb18V extracts by JA-2, the presence of a resid- housekeeping genes for this study, MIP and GAPDH. ual activity due to a MIP-like peptidase cannot be ruled out Metallopeptidases have been reported as virulence fac- in this fungus. tors in bacteria, fungi and parasites [39– 42]. In P. brasil- The hydrolysis of BK is a very important feature in the iensis, however, only recently has an enzyme been characterization of TOP-like peptidases. The cleavage of unequivocally implicated in the pathogenicity of the fun- BK by mammalian TOP and MIP has been described pre- gus, although the proteolytic activity of the molecule was viously to be at the Phe-Ser bond [15,26,32,33] and the not tested. Herná ndez et al. [4], using antisense RNA to a Pb18V cytosolic protein extract cleaved BK at the same 32-kDa protein gene member of the superfamily of hydro- site, as shown by the detection of the BK1-5 fragment. lases, showed the role of this enzyme on human epithelial © 2012 ISHAM, Medical Mycology, 50, 81–90 TOP-like metallopeptidase of P. brasiliensis 89 cell adherence and in vivo virulence of this microorganism 5 Silva Neto BR, Silva JF, Mendes-Giannini MJ, et al. The malate syn- in a murine model. Usually in the acute form of the disease thase of Paracoccidioides brasiliensis is a linked surface protein that and less so in the chronic form a Th-2-type cellular immune behaves as an anchorless adhesin. BMC Microbiol 2009; 9 : 272 – 284. 6 Donofrio FC, Calil AC, Miranda ET, et al. Enolase from Paracoc- response develops which is not protective [43– 46]. In con- cidioides brasiliensis : isolation and identifi cation as a fi bronectin- trast, healthy infected individuals or successfully treated binding protein. J Med Microbiol 2009; 58 : 706 – 713. patients develop a Th-1-type immune response with the 7 Parente JA, Salem-Izacc SM, Santana JM, et al. A secreted serine production of pro-infl ammatory cytokines, as IL-12, IL-2, protease of Paracoccidioides brasiliensis and its interactions with TNF- α and IFN- γ, associated with immune protection fungal proteins. BMC Microbiol 2010; 10 : 292 – 302. 8 Tacco BA, Parente JA, Barbosa MS, et al. Characterization of a against the fungal agent [43,46]. secreted aspartyl protease of the fungal pathogen Paracoccidioides It has been demonstrated that macrophages and dendritic brasiliensis . Med Mycol 2009; 47 : 845 – 854. cells can be activated by BK, with induction of a Th1-type 9 Carmona AK, Puccia R, Oliveira MCF, et al. Characterization of an exocellular serine-thiol proteinase activity in Paracoccidioides brasil- cytokine production, by activation of bradykinin B2 recep- Downloaded from https://academic.oup.com/mmy/article/50/1/81/989549 by guest on 30 September 2021 tors (B2R). BK-activated macrophages showed increased iensis . Biochem J 1995; 309 : 209 – 214. 10 Parente JA, Costa M, Pereira M, Soares CM. Transcriptome overview fungicidal activity [47]. Trypanosoma cruzi secretes the of Paracoccidioides brasiliensis proteases. Genet Mol Res 2005; 4 : cysteine peptidase cruzipain that generates BK which con- 358 – 371. tributes to the maturation of dendritic cells, via recognition 11 Lasdun A, Orlowski M. Inhibition of endopeptidase 24.15 slows by B2R [20,21]. Besides the effect on dendritic cells, BK the in vivo degradation of luteinizing hormone-releasing hormone. and other kinins have also been implicated in the control of J Pharmacol Exp Ther 1989; 251 : 439 – 447. 12 Molineaux CJ, Ayala JM. 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Supplementary material available online Supplementary Table 1