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FEBS Letters 581 (2007) 3882–3886

Identification and characterization of an aspartyl from Cryptococcus neoformans

Marcello Pintia, Carlotta Francesca Orsib, Lara Gibellinia, Roberto Espositoc, Andrea Cossarizzaa, Elisabetta Blasib, Samuele Peppolonib, Cristina Mussinic,* a Department of Biomedical Sciences, Chair of Immunology, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy b Department of Public Health Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy c Clinic of Infectious and Tropical Diseases, Azienda Policlinico, Via del Pozzo 71, 41100 Modena, Italy

Received 26 June 2007; accepted 3 July 2007

Available online 16 July 2007

Edited by Stuart Ferguson

encode for an aspartyl protease, usually a homodimer of a pro- Abstract Cryptococcosis, caused by Cryptococcus neoformans, is an invasive infection often occurring in AIDS patients. Potent tein of 95–125 aminoacids obtained by cleavage of a retroviral therapy against HIV, which includes protease inhibitors (PIs), precursor polyprotein [6–9]. Highly active antiretroviral ther- has beneficial effects also on opportunistic infections by patho- apy (HAART), which includes HIV protease inhibitors (PIs), gens such as C. neoformans and C. albicans. PIs inhibit growth has considerably reduced AIDS progression and mortality. of C. albicans by affecting the activity of its aspartyl . HAART has beneficial effects on some opportunistic infections We identified, cloned and sequenced a cDNA from C. neofor- by fungal pathogens, known to be major causes of morbidity mans encoding for a putative aspartyl protease (CnAP1), and and mortality in AIDS patients. In particular, we have previ- the corresponding genomic region. The gene cnap1 codifies for ously shown that HIV+ patients who experienced a cryptococ- a protein of 505 aa, with a canonical aspartyl protease structure. cal meningitis (CM) taking HAART have a lower risk of We purified the recombinant protein and analyzed its activity in relapse when the CD4 cell count increases to >100 cells/lL, the presence of PIs (Indinavir, Lopinavir, Ritonavir), but did not evidence any inhibition of protease activity. The transcriptional even in the absence of antifungal maintenance therapy [10]. level of cnap1 in C. neoformans is constant in different media. PIs such as Indinavir, Ritonavir and Lopinavir, largely used The absence of any inhibition activity by PIs suggests that other in HAART, are able to exert a direct inhibitory effect on Can- targets for PIs might exist in C. neoformans. dida albicans, and indeed fungal growth and pathogenicity are Ó 2007 Federation of European Biochemical Societies. Published profoundly affected [11]. C. albicans has numerous genes that by Elsevier B.V. All rights reserved. encode for secretory aspartyl proteases (SAPs), with important pathogenetic role in candidiasis [12–15]. Accordingly, such en- Keywords: AIDS; Opportunistic infections; Aspartyl protease; zymes, that belong to the same class of HIV proteases [6], have Protease inhibitors; C. neoformans been shown to be sensitive to PIs [15]. C. neoformans is able to produce proteases [16–20], expected to favour assimilation of nitrogen from proteins, and have been proposed as potential virulence factor(s), contributing to host 1. Introduction tissue invasion and colonisation by the pathogen. Evidence ex- ists that C. neoformans can alter its phenotype by passages in Cryptococcus neoformans is the causative agent of cryptococ- mice [21], and modify its protease production during a three- cosis, an invasive infection often occurring in HIV-infected and year-long persistence in an AIDS patient experiencing recur- AIDS patients. Other immunocompromised patients are also at rent cryptococcal meningoencephalitis [22]. high risk for cryptococcal infection [1]. Immunological compe- We have previously demonstrated that Indinavir is able to tent individuals may develop cryptococcosis, although they are directly inhibit growth and proliferation of C. neoformans in a minority compared to severely immunocompromised in vitro [23]. In particular, cryptococcal protease activity was patients [2–5]. Human infection begins with inhalation of desic- impaired in a time- and dose-dependent fashion; this phenom- cated yeasts or basidiospores followed by a local pulmonary enon was detectable either in laboratory strains or in clinical infection. Cryptococcal meningitis is the most commonly isolates. These data suggested that C. neoformans encodes for encountered life-threatening manifestation of cryptococcosis; proteases whose activity can be inhibited by PIs [23]. In this if untreated, it is always fatal. study we report the cloning and the characterization of an Aspartyl proteases, also known as acid proteases (E.C. (CnAP1) from C. neoformans, one of the pos- 3.4.23.X), are a family of proteolytic present in many sible target for PIs. unicellular and multicellular eukaryotes, including vertebrates, fungi, plants, as well as in some retroviruses, including HIV [2– 5]. Aspartyl proteases are monomeric proteins with two 2. Materials and methods domains, each containing one aspartic residue essential for enzymatic activity [6–9]. Most retroviruses, including HIV, 2.1. Fungal and bacterial strains 2.1.1. C. neoformans. We employed a C. neoformans encapsulated ATCC strain (H99) and two encapsulated strain (BPY444 and *Corresponding author. Fax: +39 059 422 2604. BPY445), derived from the wild-type fluconazole-susceptibile clinical E-mail address: [email protected] (C. Mussini). isolate strain BPY22, as detailed elsewhere [22,24,25]. C. neoformans

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.006 M. Pinti et al. / FEBS Letters 581 (2007) 3882–3886 3883 cultures were maintained in Sabouraud dextrose agar (Oxoid Hamp- 2.3.3. Real time PCR. Real time PCR was performed using UDG shire, England) by biweekly passages. The plates were kept at room Glicosylase Invitrogen Supermix, following manufacturer’s instruc- temperature. tions, on a ABI Prism 7000 Sequence Detection System (Applied Bio- 2.1.2. Escherichia coli. We employed the strains JM109 and M15/ systems, Foster City, CA, USA), using TaqMan Technology. Primers pREP4 (derived from the strain K12), grown at 37 °C in LB medium for cnap1 are CnAP1Dir (50-GGCTCCCTTTTCCAGGATCTC-30) and LB medium with kanamycin 10 lg/ml, respectively. All transfor- and CnAP1Rev (50-GACATCGTCACATGAGGCAAAG-30); Taq- mants were selected on LB media containing 100 lg/ml ampicillin for Man Probe is (50-CCTTGGCGGTTACTCGGTGGCTTCC-30). Data JM109 and 100 lg/ml ampicillin and kanamycin 10 lg/ml for M15/ were normalized to actin expression, used as a housekeeping gene. Actin pREP4, and were cultured at 37 °C overnight. was quantified using a SYBR green as fluorescent detector, with prim- ers ActinaDir (50-TCCATCCTCCACTAATCCACCTAA-30) and ActinaRev (50-CAGCACCTAGCACCCATGATC-30). Relative 2.2. Bioinformatic analysis expression of cnap1 was calculated with the ‘‘deltadelta cycle’’ method Cryptococcus sequences homologous to known proteases from C. on the basis of the difference in the threshold cycle of cnap1 and actin, albicans were searched using BLAST program available at NCBI Ser- setting expression level of control sample as 1. ver [26]. Double and multiple sequence alignments, research of con- served motifs, codon preference analysis were performed using Wisconsin Package Software. Three-dimensional structure of C. neo- formans protease was built on the basis of canonical aspartyl proteases 3. Results (proteinase A from Saccharomyces cerevisiae, endothiapepsin from Cryphonectria parasitica, from Sus scrofa and from Homo sapiens) [27–31] using Modeller software [32]. 3D images were realized Using the aminoacid sequence of SAP5 from C. albicans as a using PYMOL software [33]. query, we identified an EST clone (b1f08cn) containing a 2.2.1. Isolation of DNA and RNA. DNA was isolated from C. neo- cDNA from C. neoformans H99 with significant homology formans as previously described by bead bashing (for PCR template) in for eukaryotic aspartyl proteases. We used this sequence to a mini-bead miller (Mini-Bead beater 3110BX, Biospec Products, Bar- tlesville, OK, USA) [34]. Total RNA was isolated using RNeasy mini do a BLAST search [26] of the partially completed C. neofor- kit by QIAgen, following instructions supplied by manufacturer. mans genome sequence (November 2003 [35]) and we identified 2.2.2. Preparation of cDNA. cDNA was prepared from total RNA the genomic region containing the putative aspartyl protease using Superscript III (Gibco-BRL, Rockville, MD,USA) according to gene. manufacturer’s instructions. 2.2.3. PCR and sequencing. PCRs were performed using Expand Starting from the sequence of the EST, RACE (rapid ampli- 0 0 High Fidelity PCR System (Roche Biochemical, Mannheim, Ger- fication of cDNA ends) was used to extend the 5 and 3 ends many). Full length cDNA was obtained starting from EST sequences of the cDNA fragment. Sequencing of the genomic DNA and by RACE, using 50/30 RACE kit (Roche). The region between the 50 the 50 and 30 RACE cDNA fragments allowed us to define the 0 and 3 tags from EST was amplified by PCR using primers CnAP1Dir structure of the entire gene, which we named CnAP1. (Gen- (50-AATCCTTCCGACAGTTCC-30) and CnAP1Rev (50-ACCCC- AAATTCCCTATCTTAC-30) and sequenced with the same primers. Bank accession number DQ641261; Fig. 1 of Supplementary cDNA from C. neoformans was sequenced using BigDye 2.0 and run data). on ABI Prism 310 Genetic Analyzer. Sequences were analyzed using The gene includes three introns; all putative intron splice GCG Wisconsin Package Software. sites followed 50 and 30 consensus sequences of GTNNGY and YAG, respectively [1,36]. The position of the introns were 2.3. Protein purification confirmed by cDNA sequencing. Comparison of the DNA and C. neoformans protease was purified from E. coli JM109 transformed cDNA allowed us to identify the polyadenylation site at the with pQE80L-CnAP1 plasmid using Ni-NTA spin columns kit by nucleotide 2048 of the full cDNA; no potential polyadenyla- QIAgen (Valencia, CA, USA) following manufacturer’s instructions. In brief, E. coli/pQE80L-CnAP1 was grown at 37 °C in agitation in tion signal sequence was identified. The ORF encodes a puta- LB broth until the culture reached an absorbance of 0.6; then protease tive protein, CnAP1, of 505 residues (51.6 kDa), with a expression was induced adding IPTG 1 mM. After 0, 15 min, 30 min, predicted pK of 4.47. A homology search revealed a significant 1, 2 and 3 h, bacteria were collected, resuspended in lysis buffer degree of similarity of CnAP1 to several aspartyl proteases: (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed with lysozyme 1 mg/ml followed by four cycles of sonication pepsinogen from Aspergillus oryzae (28% identity; 46% similar- (30 s each) on ice. Lysate was loaded onto columns and washed twice ity), saccharopepsin from S. cerevisiae (29% identity; 44% sim- with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, ilarity), aspartic proteinase from Schistosoma japonicum (30% pH 8.0). 6His tagged protein was recovered with elution buffer (50 mM identity; 45% similarity), SAP5 from C. albicans (28% identity; NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). 35% similarity), among others (Fig. 2 of Supplementary data). 2.3.1. SDS–PAGE and Western blot. Total E. coli protein extract or purified protein were separated on 15% (w/v) SDS–PAGE and The aspartic residues essential for enzymatic activity were transferred to nitrocellulose membranes. Western blot was performed located at positions 85 and 278, inside two highly conserved as described [31], with little modifications. Primary antibody against aminoacidic stretches (IVLDTGSSDLWL and AAIDTGTT- 6His tag (Serotec, Raleigh, NC, USA) was diluted 1:1000 and goat LIGG). anti-mouse conjugated horseradish peroxidase (HRP) were diluted in blocking buffer (TBS-T with 3% non-fat dry milk). Membranes were The C term part of the CDS (aa 393–505) does not show any washed three times with TBS-T and antigen/antibody complexes were similarity with known proteins or domains. We have tried to detected chemiluminescence followed by autoradiography. identify consensus sequence for intracellular trafficking or 2.3.2. CnAP1 activity assay. Enzymatic activity of purified CnAP1 organelle targeting of the protein, but no similarity emerged was evaluated using EnzCheck Protease Assay Kit (Green fluores- with known consensuses of other species. cence) by Molecular Probes (Eugene, Oregon, USA) following instruc- tions supplied by the manufacturer. The reaction was carried out at The 3D models of the CnAP1 were built with a high degree 37 °C for 1 h in the presence or not of PIs (IND, LPV, RTV) or Pep- of confidence using Modeler software [29], using as a model statinA (PepA) 100 lg/ml; when assay was performed with PIs or the structures of different, well-known aspartyl proteases, such PepA, the fluorescent substrate was added after a pre-incubation of as proteinase A of S. cerevisiae, endothiapepsin from C. par- the samples with inhibitors at 37 °C for 15 min. Fluorescence was mea- sured with a Packard FluorocountTM microplate fluorometer. Values asitica, renin and pepsin from H. sapiens Li, 2000 #151; are indicated as relative activity respect to control (without any inhib- Coates, 2003 #109; Baldwin, 1993 #148; Fujinaga, 1995 itor) set arbitrarily as 1. #147; Rahuel, 1991 #149; Sielecki, 1989 #110. 3D homology 3884 M. Pinti et al. / FEBS Letters 581 (2007) 3882–3886 model building was possible only for the conserved part of the protein (aa 45–388), whereas the structure of the C term, un- known domain could not be determined (Fig. 1). Results, very similar for both algorithms, indicate that an almost complete structural homology exists between CnAP1 and other aspartyl proteases in the interface of the catalytic domains and in the . The large part of non-conservative substitutions re- gards amino acids located on the outer surface of the protein, whereas the inner core of the is mostly identical or sim- ilar to canonical, monomeric aspartyl proteases. In particular, the sequence and the relative position of the residues necessary for catalytic activity is completely conserved. Then we have cloned the entire CDS in pQE80L vector, so adding an in-frame 6His tag at the N terminus of the protein. We expressed the recombinant protein in E. coli JM109 carry- ing the pQE80L-CnAP1 plasmid after induction with IPTG for 3 h. Samples were collected at different times to evidence accu- mulation of recombinant protein. As shown by Western blot Fig. 2. (A) Western blot of CnAP1 expression in Escherichia coli analysis (Fig. 2), a band with an apparent molecular weight JM109 plasmid pQE80L-CnAP1 after induction with 1 mM IPTG at of 49 kDa appears after 15 min of induction with IPTG, and different times. The band corresponding to recombinant CnAP1 is accumulates until 3 h after adding IPTG to the culture. To- indicated by a black arrow. (B) Western blot of CnAP1, cloned in E. coli with plasmid pQE80L, purified with column affinity in native gether with the major, full length form of the protein, many conditions. The band corresponding to recombinant CnAP1 is other proteins with lower molecular weight, presumably trun- indicated by a black arrow. WF, wash fraction with 10 mM imidazole; cated form of CnAP1, accumulated in the cell. The protein E, eluate in the presence of 200 mM imidazole. was purified under denaturing and native conditions using nickel affinity chromatography, using lysate obtained from trol, at two different concentrations (2.5 and 25 lM). As short induction times (15 and 30 min), to avoid premature ter- shown in Fig. 3, we could not evidence any inhibition of pro- mination. Western blots show that the only form of recombi- tease activity for all of the three drugs, at both concentrations, nant protein purified is the full length form. The recombinant whereas a broad range inhibitor of aspartyl proteases like Pep- protein was purified at low levels, probably for the incomplete statin A reduced enzymatic activity of about 87% respect to exposure of the tag in native conditions. We could not avoid the control. copurification of other proteins together with 6His CnAP1, de- In order to exclude that the presence of the 6His tag at the N spite changes in washing and elution conditions. However, no term of the enzyme could interfere with the activity of the en- protease activity was detected in the column eluate from non- zyme, and so preclude the inhibitory activity of the drugs, we stimulated samples, indicating that none of the copurified pro- cloned the CDS of CnAP1 in the vector pQE30-Xa, produced teins is an active protease. Test of protease activity indicates and purified the recombinant enzyme in E. coli M15/pREP4. that CnAP1 has a maximum activity at pH 5.0. After proteolytic digestion with Factor Xa, which eliminates We have analyzed the activity of the enzyme in the presence the 6His tag, the enzyme was tested in the presence of PIs; of three different PIs (IND, LPV, RTV), compared to the con- no inhibition of its activity was observed (data not shown). Finally, we wondered if cnap1 transcription could be modu- lated by the presence of proteins in the environment where C. neoformans is grown. We have analyzed the transcriptional level of cnap1 in C. neoformans, cultured in a rich medium (YEPD) and in a minimal medium where proteins were the sole energy source. As shown in Fig. 4, the expression level of cnap1 is maintained at similar levels, indicating that pres- ence of proteins as sole energy source does not influence tran- scription of the gene. Furthermore, cnap1 expression seems depending on the growth phase, with higher expression in sta- tionary phase than in exponential phase.

4. Discussion

We have cloned and characterized an aspartyl protease gene of C. neoformans, giving for the first time the genetic evidence that this fungus bears gene(s) encoding for this kind of prote- ases. The C term portion of this protein does not show any Fig. 1. Three-dimensional ribbon model of CnAP1, built on the basis similarity with known proteins, and so its role remains unclear. of 3D structure of a known aspartyl proteases (proteinase A from Saccharomyces cerevisiae, endothiapepsin from Cryphonectria parasit- We could suppose that this region, like many other domains of icafrom, renin from Homo sapiens, pepsin from Sus scrofa). The proteases or catabolic enzymes, represents an inhibitor domain catalytic, conserved Asp residues are indicated in blue. that is cleaved to activate the enzyme, produced as a zymogen, M. Pinti et al. / FEBS Letters 581 (2007) 3882–3886 3885

1.50

1.00

0.50 Relative activity 0 Control 2.5 μM 25 μM 2,5 μM 25 μM 2,5 μM 25 μM PepA 100μ g/ml IND LPV RTV

Fig. 3. Relative enzymatic activity of CnAP1 in the presence or not of PIs at the indicated concentrations. Relative activity is referred to control (sample without any drug), arbitrarily set as 1; results are indicated as means ± S.E.M. of three independent experiments. IND, Indinavir; RTV, itonavir; LPV, lopinavir; PepA, Pepstatin A.

interesting to note that, compared to C. albicans, C. neofor- 1.75 Med mans has got a lower number of genes encoding for this class 1.50 YEPD of protease; we can assume that, in the environment where 1.25 Cryptococcus has a strong tropism, (secreted?) proteases do not play the same, crucial role that play in the case of Candida. 1.00 Another possible explanation is that since purified CnAP1 0.75 we have analyzed is produced in E. coli, it does not undergo post translational modifications. Analysis of the sequence 0.50 has evidenced the presence of many putative glycosylation sites in the C-term portion of the enzyme. We can suppose that in

Relative expression 0.25 the absence of such modifications, the enzyme does not have 0 the native 3D structure, so changing the accessibility to the 0 h 6 h 24 h drug, and drug–protein interaction. Fig. 4. Transcriptional levels of cnap1 gene in Cryptococcus neofor- As far as transcriptional regulation is concerned, we could mans grown in YEPD and minimal medium (Med) for indicated times. not evidence any significant modulation of cnap1 expression Relative expression is referred to time 0 (before induction) set with different media, suggesting that C. neoformans has no di- arbitrarily as 1. rect way to regulate the transcription on the basis of the com- ponent of the medium. However, the observation that cnap1 or a sequence for intracellular trafficking. Since the mecha- transcription is increased when C. neoformans reaches the sta- nisms by which proteins are distributed inside the Cryptococ- tionary phase, regardless of the medium considered, indicates cus cell are not well known, it is not possible to identify that this gene is effectively regulated and that its protease activ- consensus sequences for organelle targeting or secretion. The ity has probably a role during the lag phase. The discrepancy presence of stretches of Ser residues could suggest that this between the ability of PIs to inhibit growth of C. neoformans part of CnAP1 is a target for glycosylation. We are now trying and the absence of inhibitory activity of this class of drugs to express the enzyme in S. cerevisiae and in human, cultured on CnAP1 indicates that further studies are needed to identify cells to evaluate a possible glycosylation, and contemporarily other targets of PIs in this opportunistic pathogen and we are producing a truncated form of the enzyme, in order better clarify the role of PIs in the control of opportunistic to evaluate differences enzymatic activity, and thus clarify infections. the eventual, inhibitory activity of this domain. Analysis of the 3D structure of the enzyme, possible only for Acknowledgements: This work was supported by the Grant 50F.25 of the N-term portion of the enzyme (aa 45–388) clearly indicates Istituto Superiore di Sanita` (Rome, Italy) to C.M. Dr. Maria Scarselli that CnAP1 is a canonical aspartic protease, with two homo- is kindly acknowledged for assistance in 3D modeling. logous domains each containing an Asp residue, located face-to-face in the centre of the enzyme. The relative position Appendix A. Supplementary data of the essential residues is perfectly conserved. We could not evidence any inhibition of protease activity by Supplementary data associated with this article can be HIV PIs even if we have previously shown that IND, a drug found, in the online version, at doi:10.1016/j.febslet.2007. belonging to PI class, can directly inhibit C. neoformans 07.006. growth [25]. There are several possible explanations for this re- sult. The simplest is that CnAP1 is not the direct target of these drugs, and that other aspartyl proteases existing in C. neofor- References mans represent the real target. We have scanned the C. neofor- [1] Casadevall, A. and Perfect, J.R. (1998) Cryptococcus neoformans. mans cDNA database again, finding just another one encoding Washington, DC. p. 542. for a putative aspartyl protease. This gene, that we have named [2] Kervinen, J., Tormakangas, K., Runeberg-Roos, P., Guruprasad, cnap2, is now under cloning and investigation. However, it is K., Blundell, T. and Teeri, T.H. (1995) Structure and possible 3886 M. Pinti et al. / FEBS Letters 581 (2007) 3882–3886

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