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Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein
Experimental Parasitology 119 (2008) 358–363
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein Andrés J. Gutierrez-Escobar, Aylan Farid Arenas, Yanet Villoria-Guerrero, Jonathan M. Padilla-Londoño, Jorge Enrique Gómez-Marin * Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia
a r t i c l e
i n f o
Article history: Received 1 March 2007 Received in revised form 13 March 2008 Accepted 18 March 2008 Available online 23 March 2008 Index Descriptors and Abbreviations: Toxoplasma Nitric oxide synthase Griess assays Baculovirus Colorimetric assay Protozoa Bioinformatics
a b s t r a c t Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of L-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis–Menten kinetics, with a Km for L-arginine of 2.3 mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Nitric oxide (NO) is a second messenger molecule that participates in a plethora of cellular and systemic physiological conditions, e.g., regulation of vascular tone, neuronal transmission, or in antitumoral or antimicrobial activities (Moncada et al., 1991). NO in mammals is produced from L-arginine by three isoforms of nitric oxide synthases (NOS) that can have natural or inducible activity. It has been also demonstrated that invertebrates, such as certain insect species (Weiske and Wiesner, 1999), molluscs (Huang et al., 1997), nematodes such as Ascaris suum (Bascal et al., 1995), Dirofilaria immitis (Kaiser et al., 1998) or Brugia species (Pfarr and Fuhrman, 2000) and bacteria (Adak et al., 2002) are able to produce NO and have their own NOS-like genes. In this context little is known about protozoan NOS. Different studies show that Toxoplasma (Gomez Marin, 2000) in common with other protozoa such as Tetrahymena (Christensen, 1996), Trypanosoma (Paveto et al., 1995), Entamoeba (Hernandez-Campos et al., 2003) and Plasmodium (Ghigo et al., 1995) has its own nitrite production that would reflect natural NOS activity (producing 2–6 lM of nitrites). Genestra et al. (2003) have also shown that Leishmania produce levels of nitrite similar to those found in Toxoplasma. Gutierrez-Escobar and Gómez-Marin (2005) iden-
* Corresponding author. Fax: +57 67 460168. E-mail address: [email protected] (J.E. Gómez-Marin). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.03.008
tified the first protozoal genomic sequence that included one putative NOS motif signature and also demonstrated that it was transcriptionally active. This was a strong argument to believe that nitrites in Toxoplasma would be derived from a NOS enzyme that could be part of a L-arginine pathway. As a consequence of this previous work, we cloned the Toxoplasma sequence containing the universal signature motif NOS (oxygenase domain) into a recombinant baculovirus system in order to demonstrate that Toxoplasma has a functional NOS-like protein. 2. Materials and methods 2.1. Parasites Female Swiss ICR mice (Universidad Nacional, Bogota, Colombia) were inoculated with Toxoplasma gondii RH strain. Tachyzoites were recovered from the peritoneal cavity 3–4 days later by instilling 5 ml sterile 0.9% NaCl solution with antibiotics (penicillin 100 U/ml and streptomycin 100 lg/ml). Tachyzoites were isolated by centrifugation twice at 200g for 10 min. The pellets were resuspended in RPMI medium and filtered through 3 lm pore size polycarbonate membrane (Nucleopore, Cambridge). Cell and parasite viability was tested with the Trypan blue exclusion test (0.4% solution) and only samples with 95% or more viable parasites were used.
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2.2. Cell and baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) and its recombinant virus were grown in Spodoptera frugiperda (Sf9) cells in a TNM-FH insect medium (Clontech, CA, USA) supplemented with antibiotics (penicillin 100 U/ml and clamoxicillin 100 lg/ml).
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days 8 and 15 and serum was obtained at day 45. ELISA was performed to determine the titers of polyclonal antibody sera and fluorescent staining was performed with 1/10 and 1/50 dilutions of antisera. Controls was undertaken using preimmune antisera and secondary antibody without polyclonal antibodies and testing with two recombinant proteins (Toxoplasma metalloprotease and Entamoeba histolytica cysteine protease) obtained by the His tagged procedure.
2.3. Cloning of the Toxoplasma NOS gene 2.7. NO synthase assay The template RNA for RT-PCR was extracted from tachyzoites of the T. gondii RH strain as described previously (Gutierrez-Escobar and Gómez-Marin, 2005). Two oligonucleotide primers: Tgnos1: AGGCCT-CTGCTTGCCGTTTGTTTCG and Tgnos2: CGCCGGCGGCACACGCTCAACTAATTAC including the restriction sites Stu1 and Not1 (underlined in the primer sequences) were used to amplify the Toxoplasma NOS gene by RT-PCR assay as described previously (Gutierrez-Escobar and Gómez-Marin, 2005) and then the product was recovered from agarose. The transfer vector pAcHLTA was digested with Stu1 and Not1 restriction enzymes. The purified Toxoplasma NOS PCR product amplification was ligated into the Stu1 and Not1 sites of the baculovirus transfer vector pAcHLT-A (Becton–Dickinson, Franklin Lakes, NJ, USA). The resulting plasmid was designated pAcHLT-A-NOS.
NO synthesis by Toxoplasma NOS recombinant protein of T. gondii was measured in a microplate assay for nitrite based on the Griess reaction (Stuehr and Marletta, 1985) and using the Nitric Oxide Synthase Assay Kit (Bioxytech, Foster City, USA) as recommended by the manufacturer. The reaction was run at room temperature for 40 min. Residual NADPH, which could interfere with the colorimetric assay, was removed at the end of the incubation by adding 40 U/ml of lactate dehydrogenase. The stability of the protein was determined by incubating the protein for 10 min at a range of temperatures between 0 and 100 °C and then the NOS assay was performed. The pH range for the recombinant NOS was evaluated using sodium acetate buffer at pH 4.5, potassium phosphate buffer at pH 7.0 and 7.5, and Tris–HCl buffer at pH 9.5 in the colorimetric assay.
2.4. Construction of recombinant baculovirus and DNA sequencing 2.8. Statistics Sf9 cells were cotransfected with the recombinant transfer vector pAcHLT-A-NOS and linear AcNPV viral DNA (Becton–Dickinson, Franklin Lakes, NJ, USA) following the manufacturer instructions for the Baculovirus Expression and Purification Kit (Clontech, California, USA). After 4 days incubation at 27 °C, the cells and supernatant containing the recombinant virus were harvested and a recombinant baculovirus was obtained. A recombinant transfer vector pAcHLT-XyIE was used as a control virus. Sequencing of recombinant plasmids was performed from Toxoplasma NOS PCR products. Specific bands were cut from low-melt agarose gels, followed by recovery using the Wizard PCR Prep kit (Promega, WI). Sequencing was done by using the ABIPRISM model 3100 version 3.7 at the Wadsworth Genetics Institute facility on purified DNA by using 2 pmol of the Tgnos1 and Tgnos primers. 2.5. Recombinant Toxoplasma NOS protein purification and electrophoretic analysis The sf9 cells were treated with lysis buffer and the supernatant was cleared by centrifugation. Later, they were added to a vial with prewashed Ni–NTA (Ni2 + nitrilotriacetate) beads (Clontech, CA, USA). The mixture was incubated at 4 °C for 1 h. The beads were collected, washed with lysis buffer and then, the Toxoplasma NOS protein that was bound to the beads, was eluted with lysis buffer containing 250 mM imidazole according to the manufacturer instructions. Afterwards, the protein eluted was quantified by the Bradford method. Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (trycine SDS–PAGE) was performed as described by Schägger (2006).
All data are expressed as means ± SEM from three experiments for NOS activity and in duplicate for kinetic, temperature and pH experiments. 3. Results 3.1. Cloning and bioinformatics analysis of Toxoplasma NOS-like gene The product of reverse transcription was amplified revealing a band with the expected size (237 pb) for a Toxoplasma NOS-like gene (Fig. 1). No amplification product was observed in the negative control, indicating that there was no contamination with genomic DNA. The similar sizes of bands obtained both in PCR and in RT-PCR reactions indicated that Toxoplasma NOS-like gene consisted of a single exon and this was further confirmed sequencing the cloning amplification products. During cloning, we used primers with restriction sites obtaining similar bands (Fig. 1). The Toxoplasma NOS product was purified from RT-PCR (Fig. 1, Lane 3) and then ligated into the baculovirus transfer vector pAcHLT-A and cloned into Escherichia coli DH5a strain. The recombinant vector was used in a Toxoplasma NOS gene PCR assay (Gutierrez-Escobar and Gómez-Marin, 2005) and sequenced (Fig. 2). The analysis of the sequence by using basic local alignment tool (BLAST) in ToxoDB (http://toxoDB.org) showed 100% of identity with our previously reported Toxoplasma NOS gene listed in the ToxoDB database (Fig. 2B). Furthermore, the Toxoplasma NOS gene was not present in the genome of the Sf9 culture cells after
2.6. Animal antisera and fluorescence staining One New Zealand White rabbit (2 kg) was housed, fed and handled in accordance with the recommendations made by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA). One hundred fifty micrograms of purified NOS protein emulsified in hydroxide aluminum adjuvant was inoculated intramuscularly at four sites. The same amount of immunogen mixed with aluminum hydroxide was inoculated as a booster on
Fig. 1. Toxoplasma NOS gene RT-PCR amplification in Toxoplasma gondii. Lane 1, positive kit control; lane 2, RT-PCR of DNase-treated total T. gondii RNA using the primers without restriction sites; lane 3, the RT-PCR of DNase-treated total T. gondii RNA using the primers with restriction sites; lane 4, negative control.
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Fig. 2. PCR assay of Toxoplasma NOS gene from pAcHLT-NOS. (A) Lanes 1 and 2, PCR products from the recombinant transfer vector pAcHLT-A-NOS by using primers with restriction sites; lane 3, PCR product from recombinant transfer vector pAcHLT-A-NOS by using primers without restriction sites; lane 4, negative control. (B) BLAST analysis at ToxoDB database of the sequence obtained from the Toxoplasma NOS PCR product (lane 3). (C) Clustal multiple alignment of the oxygenase domains of Drosophila melanogaster (NP_5233541), Deinococcus radiodurans (Q9RR97) and Bacillus halodurans (NP_241475) with the Toxoplasma NOS-like recombinant deduced aminoacid sequence.
the cotransfection process (Fig. 3). The recombinant NOS gene sequence was deposited in the GenBank (Accession No. EF452680). The deduced aminoacid sequence (R P L L A V C F V S D F Y S L S LLHFASVPFHESDGCVGRSHWLPGKHANYVKP A G A R K R P E V G C R S S C L L R S V C C D I L S P V R T R G N Stop L S V C) showed that the NOS signature motif was conserved (GCVGRS). The deduced aminoacid sequence was searched against the ORF database at ToxoDB.org (consulted February 9, 2008) and matched with a sequence (ORF 1b-4) at the chromosome 1b between pairs bases: 1,352,191–1,352,429. A promoter region is found at a distance of 2373 bp in the region between nucleotides 1,354,800 and 1,356,600 (Mathieu Gissot, personal communication). No similar sequences were found in PlasmoDB with this ORF. Interproscan analysis at www.expasy.org looking for protein domains of the ORF sequence indicates a signal peptide region, between amino acids 1–28, and the NOS signature motif, between amino acids 29–35. Additionally, the recombinant sequence was aligned against the ORF sequence of ToxoDB and 79 of the 81 residues were identical. Only the first two residues (R and P) of the recombinant protein are not present at the ORF at ToxoDB database
(shown in bold); this amino acids come from the restriction sites artificially introduced for cloning purposes into the baculovirus. An important characteristic of the NOS-like coding sequence is the presence of an alternative codon (CTG, leucine) at the beginning of the open reading frame. The theoretical molecular weight for the recombinant sequence was estimated to be 8.9 kDa and to have a pI of 9.3. 3.2. Biochemical and enzymatic analysis of recombinant Toxoplasma NOS-like protein We obtained three fractions of Ni–NTA-purified Toxoplasma NOS-like protein, with a concentration of 339, 203 and 455 lg of protein by ml. The activity of the purified recombinant protein in the NOS colorimetric assay in each of the fractions was 21.8 ± 0.9; 5.2 ± 0.2 and 3.7 ± 1.01 nmol/min/ml per lg of protein, respectively. Subsequent experiments were performed with the fraction with the highest activity. Activity was reduced only by 33% in absence of NADPH and by 66% when cofactors were absent and it was nearly inhibited when L-arginine was absent (Table 1).
Fig. 3. Sf9 culture cell. (A) Healthy Sf9 cells cultured in TNM-FH medium before cotransfection process. (B) Toxoplasma NOS PCR control in genomic DNA of Sf9 cell. 1, Positive control (Toxoplasma gondii DNA); 2, Toxoplasma NOS PCR in genomic DNA of Sf9 cells; 3, negative control.
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Tricine–SDS–PAGE with the fraction with the highest production of nitrites showed an unique band with a molecular weight of 11.6 kDa, as estimated by calculating the relative electrophoretic mobility (Fig. 4). Kinetic analysis of the purified enzyme was made using a range of L-arginine concentrations from 0.5 to 40 mM. The kM for the recombinant protein, as estimated by the double reciprocal plot of kinetic analysis, was 2.3 mM L-arginine (Fig. 5). The temperature for a maximum enzymatic activity was measured by
Table 1 Nitric oxide synthase activity based on Griess colorimetric reaction Nitrites production per microgram of protein (nmol/ml/min) Condition Condition Condition Condition Control
A B C D
21 ± 1.07 7 ± 1.4 14.6 ± 0.97 0.7 ± 0.9 0±0
Data are means ± SEM. (A) Purified recombinant Toxoplasma NOS-like protein + Larginine + NADPH + Cofactors (BH4 + FAD + FMN + Hem + Calmodulin) + Nitrite reductase. (B) Without Cofactors (BH4 + FAD + FMN + Hem + Calmodulin). (C) Without NADPH. (D) Without L-arginine. Control: Distilled water with L-arginine + Cofactors + Nitrate reductase.
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incubating the protein during 10 min at 0, 25, 37, 60 and 100 °C. The enzyme showed a maximum activity at 25 °C and lost about 60% of its activity when it was incubated at 37 °C (Fig. 6a). The optimum pH of the recombinant NOS occurred between 7.0 and 7.5 using potassium phosphate buffer (Fig. 6b). Activity was maximal after 40 min of incubation of enzyme and substrate (Fig. 6c). 3.3. Immunodetection of NOS-like protein The polyclonal antibody was first tested in an ELISA by using the His- tagged recombinant proteins for recombinant Toxoplasma NOS-like, metalloprotease or Entamoeba histolytica cysteine protease as an antigens coated to the plate wells. The absorbance of the preimmune serum was 0.06 ± 0.00 and after 4 weeks of inoculation the absorbencies for recombinant NOS-like was 0.44 ± 0.05, for recombinant Toxoplasma metalloprotease, 0.08 ± 0.0 and for recombinant Entamoeba histolytica cysteine protease, 0.08 ± 0.0. The serum obtained after 4 weeks of post-immunization was used in an immunofluorescence assay at dilutions of 1/50 and 1/10. A specific diffuse labeling of the entire body of the tachyzoites was observed (Fig. 7). 4. Discussion
Fig. 4. Tricine–SDS–PAGE of the Toxoplasma NOS recombinant protein. Arrows signals the bands for the Toxoplasma NOS recombinant protein and the bovine lactoalbumin molecular weight control marker (14 kDa).
Previously we identified the Toxoplasma NOS putative gene sequence and determined that the NOS sequence in Toxoplasma is a primitive one, as could be inferred from the phylogenetic analysis with the NOS sequences currently known (Gutierrez-Escobar and Gómez-Marin, 2005). Here, we have performed an initial preliminary biochemical characterization of the recombinant Toxoplasma NOS-like protein obtained from this genomic sequence. The enzymatic assays showed that the recombinant protein is able to produce nitrites from L-arginine. Antibodies against this recombinant protein specifically labeled Toxoplasma tachyzoites, confirming that it is a protein present in the parasite. Nitric oxide synthase enzymes in mammals are composed of oxygenase and reductase domains. However, bacterial NOS homologs, in the same manner as the Toxoplasma NOS-like pro-
Fig. 5. Kinetic analysis of the recombinant Toxoplasma nitric oxide synthase-like protein.
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Fig. 6. Nitrites production of the recombinant Toxoplasma nitric oxide synthase-like protein: (a) Effect of temperature on stability of the enzymatic activity of the protein. (b) Effect of modifications on pH buffer during nitrites assay. (c) Effect of different incubation time of the protein and substrate for the colorimetric assay.
Fig. 7. Immunofluorescence assay on tachyzoites with rabbit polyclonal antibodies anti-recombinant Toxoplasma NOS-like protein: (a) Control with preimmune rabbit serum 1/10. (b) 4 week post-immune rabbit serum at a dilution 1/50. (c) 4 week post-immune rabbit serum at a dilution 1/10.
tein, do not have the reductase domain. Thus, the critical question is how electrons are being donated to the oxygenase domain. In a structure modeling for bacterial NOS without reductase domains, Zemojtel et al. (2003) proposed that the heme-binding loops as well as the heme groups fit well in the cleft present in the vicinity of the NOS oxygenase N-terminal region. This model is therefore a possible configuration of a docking mode for bacterial NOS oxygenase domain which would not be possible for eukaryotic NOSs with an extended N-terminal part. This could explain how the oxygenase domain catches electrons without a reductase domain. Our kinetic measurements show that Toxoplasma NOSlike protein is not as efficient as mammalian NOS; therefore the presence of the two domains being expressed in one amino acid chain should allow for a more efficient NO catalysis in mammals. Alternatively, in vivo, Toxoplasma NOS-like protein could interact with the product of other genes with reductase like functions that would increase the NOS activity, as it has been proposed for bacteria (Zemoj-
tel et al., 2003). Some prokaryote NOS proteins, such as those from Deinococus, bound arginine and cofactor H4B, and had a normal heme environment. This despite missing N-terminal structures that bind Zn, and the dihydroxypropyl side chain of H4B, which help form an active dimer in mammalian NOS (Adak et al., 2002). Additionally the size of the recombinant Toxoplasma NOS-like protein is very small (11.6 kDa), similar to the 26 kDa NOS protein reported in E. histolytica (Hernandez-Campos et al., 2003) but smaller than the 40 kDa prokaryotic NOS protein in Bacillus subtilis (Adak et al., 2002) and that of 52 kDa in Nocardia species (Chen and Rosazza, 1995). The NOS gene sequence in Entamoeba is still unknown. The NOS proteins in mammals have been reported to be about 130 kDa (Moncada et al., 1991) and to be 160 kDa in arthropods such as Drosophila (Müller, 1994). The differences in the molecular weights in the NOS family therefore show an evolutionary divergence. Nitrite production by Toxoplasma NOS recombinant protein was approximately 21 nmol/min per lg of protein, more higher than that
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reported for E. histolytica NOS activity (5 nmol/min per lg of protein) (Hernandez-Campos et al., 2003) Our bioinformatic and biochemical results, suggest that T. gondii expresses a protein which is different from NOS isoforms found in mammalian cells and invertebrates but very similar to Deinococcus, and other bacterial NOS, which only contained an oxygenase domain. Previous observations indicated that T. gondii is an obligatory auxotroph for cytoplasmatic host cell L-arginine, because it does not have the enzymatic machinery for its synthesis (Fox et al., 2004). Furthermore, parasite cultures without L-arginine show an increase in the tachyzoites to bradyzoites interconversion (Fox et al., 2004). The present report is a first approach to analyze the characteristics of the recombinant Toxoplasma NOS-like protein and future work will study the localization and trafficking of the protein and characterize its catalytic parameters in both multiple and single turnover reactions. We also intend to study of inhibitors and analogs of L-arginine and address the role of calcium and others cofactors for Toxoplasma NOS activity. NO synthesis by a Toxoplasma NOS-like protein is now established, but a number of related issues remain to be explored. These include the crystal structure, the native redox partner(s), what Noxide product is generated during steady state synthesis, what conditions induce expression of these proteins, and whether allosteric factors exist that modulate kinetic parameters of the enzyme. Further research should therefore provide a deeper understanding of NOS structure/function and the evolutionary consequence of the NOS gene and its potential as chemotherapeutic target. Acknowledgments This work was supported by Colciencias, Grant No. 1113-0418247. We thank to Drs. Vern Carruthers and My-Hang Huynh from the Department of Microbiology of the University of Michigan for providing material, technical advice and critical discussion of the results and to Dr. Carlos Saavedra and Jason Isabelle, from the Wadsworth Institute, NY, for helping in sequencing recombinant DNA plasmids. References Adak, S., Aulak, K.S., Stuehr, J., 2002. Direct evidence for nitric oxide production by a nitric-oxide synthase protein from Bacillus subtilis. The Journal of Biological Chemistry 277, 16167–16171.
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Bascal, Z.A., Montgomery, A., Holden-Dye, L., Williams, R.G., Walker, R.J., 1995. Histochemical mapping of NADPH diaphorase in the nervous system of the parasitic nematode Ascaris suum. Parasitology 110, 625–637. Chen, Y., Rosazza, J., 1995. Purification and characterization of nitric oxide synthase (NOSNoc) from a Nocardia species. Journal of Bacteriology 177, 5122–5128. Christensen, S.T., 1996. Cell death, survival and proliferation in Tetrahymena thermophila. Effects of insulin, sodium nitroprusside, 8-bromo cyclic GMP, NG methyl-L-arginine and methylene blue. Cell Biology International 10, 653– 666. Fox, B.A., Gigley, J.P., Bzik, D.J., 2004. Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation. International Journal for Parasitology 34, 323–331. Genestra, M., de Souza, W.J., Cysne-Finkelstein, L., Leon, L.L., 2003. Comparative analysis of the nitric oxide production by Leishmania sp.. Medical Microbiology and Immunology 192, 217–223. Ghigo, D., Todde, R., Ginsburg, H., Costamagna, C., Gautret, P., Bussolino, F., Ulliers, D., Giribaldi, G., Deharo, E., Gabrielli, G., Pescarmona, G., Bosia, A., 1995. Erythrocyte stages of Plasmodium falciparum exhibit a high nitric oxide synthase (NOS) activity and release an NOS-inducing soluble factor. Journal of Experimental Medicine 182, 677–688. Gutierrez-Escobar, A.J., Gómez-Marin, J.E., 2005. Toxoplasma gondii: Identification of a putative nitric oxide synthase motif DNA sequence. Experimental Parasitology 111, 211–218. Gomez Marin, J.E., 2000. No NO production during human Toxoplasma infection. Parasitology Today 16, 131. Hernandez-Campos, M.E., Campos-Rodrıguez, R., Tsutsumi, V., Shibayama, M., Garcıa-Latorre, E., Castillo-Henkel, C., Valencia-Hernandez, I., 2003. Nitric oxide synthase in Entamoeba histolytica: its effect on rat aortic rings. Experimental Parasitology 104, 87–95. Huang, S., Kerschbaum, H.H., Engel, E., Hermann, A., 1997. Biochemical characterization and histochemical localization of nitric oxide synthase in the nervous system of the snail, Helix pomatia. Journal of Neurochemistry 69, 2516–2528. Kaiser, L., Geary, T.G., Williams, J.F., 1998. Dirofilaria immitis and Brugia pahangi: filarial parasites make nitric oxide. Experimental Parasitology 90, 131–134. Moncada, S., Palmer, R.M., Higgs, E.A., 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews 43, 109–142. Müller, U., 1994. Ca2+/calmodulin-dependent nitric oxide synthase in Apis mellifera and Drosophila melanogaster. European Journal of Neurosciences 6, 1362–1370. Paveto, C., Pereira, C., Espinosa, J., Montagna, A.E., Farber, M., Esteva, M., Flawia, M.M., Torres, H.N., 1995. The nitric oxide transduction pathway in Trypanosoma cruzi. Journal of Biological Chemistry 270, 16576–16579. Pfarr, K.M., Fuhrman, J.A., 2000. Brugia malayi: localization of nitric oxide synthase in a lymphatic filarid. Experimental Parasitology 94, 92–98. Schägger, H., 2006. Tricine–SDS–PAGE. Nature Protocols 1, 16–22. Stuehr, D.J., Marletta, M.A., 1985. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccaride. Proceedings of the National Academy of Sciences of the United States of America 82, 7738–7742. Zemojtel, T., Wade, R.C., Dandekar, T., 2003. In search of the prototype of nitric oxide synthase. FEBS Letters 554, 1–5. Weiske, J., Wiesner, A., 1999. Stimulation of NO synthase activity in the immunecompetent lepidopteran Estigmene acraea hemocyte line. Nitric Oxide 3, 123– 131.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein Andrés J. Gutierrez-Escobar, Aylan Farid Arenas, Yanet Villoria-Guerrero, Jonathan M. Padilla-Londoño, Jorge Enrique Gómez-Marin * Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia
a r t i c l e
i n f o
Article history: Received 1 March 2007 Received in revised form 13 March 2008 Accepted 18 March 2008 Available online 23 March 2008 Index Descriptors and Abbreviations: Toxoplasma Nitric oxide synthase Griess assays Baculovirus Colorimetric assay Protozoa Bioinformatics
a b s t r a c t Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of L-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis–Menten kinetics, with a Km for L-arginine of 2.3 mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate. Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction Nitric oxide (NO) is a second messenger molecule that participates in a plethora of cellular and systemic physiological conditions, e.g., regulation of vascular tone, neuronal transmission, or in antitumoral or antimicrobial activities (Moncada et al., 1991). NO in mammals is produced from L-arginine by three isoforms of nitric oxide synthases (NOS) that can have natural or inducible activity. It has been also demonstrated that invertebrates, such as certain insect species (Weiske and Wiesner, 1999), molluscs (Huang et al., 1997), nematodes such as Ascaris suum (Bascal et al., 1995), Dirofilaria immitis (Kaiser et al., 1998) or Brugia species (Pfarr and Fuhrman, 2000) and bacteria (Adak et al., 2002) are able to produce NO and have their own NOS-like genes. In this context little is known about protozoan NOS. Different studies show that Toxoplasma (Gomez Marin, 2000) in common with other protozoa such as Tetrahymena (Christensen, 1996), Trypanosoma (Paveto et al., 1995), Entamoeba (Hernandez-Campos et al., 2003) and Plasmodium (Ghigo et al., 1995) has its own nitrite production that would reflect natural NOS activity (producing 2–6 lM of nitrites). Genestra et al. (2003) have also shown that Leishmania produce levels of nitrite similar to those found in Toxoplasma. Gutierrez-Escobar and Gómez-Marin (2005) iden-
* Corresponding author. Fax: +57 67 460168. E-mail address: [email protected] (J.E. Gómez-Marin). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.03.008
tified the first protozoal genomic sequence that included one putative NOS motif signature and also demonstrated that it was transcriptionally active. This was a strong argument to believe that nitrites in Toxoplasma would be derived from a NOS enzyme that could be part of a L-arginine pathway. As a consequence of this previous work, we cloned the Toxoplasma sequence containing the universal signature motif NOS (oxygenase domain) into a recombinant baculovirus system in order to demonstrate that Toxoplasma has a functional NOS-like protein. 2. Materials and methods 2.1. Parasites Female Swiss ICR mice (Universidad Nacional, Bogota, Colombia) were inoculated with Toxoplasma gondii RH strain. Tachyzoites were recovered from the peritoneal cavity 3–4 days later by instilling 5 ml sterile 0.9% NaCl solution with antibiotics (penicillin 100 U/ml and streptomycin 100 lg/ml). Tachyzoites were isolated by centrifugation twice at 200g for 10 min. The pellets were resuspended in RPMI medium and filtered through 3 lm pore size polycarbonate membrane (Nucleopore, Cambridge). Cell and parasite viability was tested with the Trypan blue exclusion test (0.4% solution) and only samples with 95% or more viable parasites were used.
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2.2. Cell and baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) and its recombinant virus were grown in Spodoptera frugiperda (Sf9) cells in a TNM-FH insect medium (Clontech, CA, USA) supplemented with antibiotics (penicillin 100 U/ml and clamoxicillin 100 lg/ml).
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days 8 and 15 and serum was obtained at day 45. ELISA was performed to determine the titers of polyclonal antibody sera and fluorescent staining was performed with 1/10 and 1/50 dilutions of antisera. Controls was undertaken using preimmune antisera and secondary antibody without polyclonal antibodies and testing with two recombinant proteins (Toxoplasma metalloprotease and Entamoeba histolytica cysteine protease) obtained by the His tagged procedure.
2.3. Cloning of the Toxoplasma NOS gene 2.7. NO synthase assay The template RNA for RT-PCR was extracted from tachyzoites of the T. gondii RH strain as described previously (Gutierrez-Escobar and Gómez-Marin, 2005). Two oligonucleotide primers: Tgnos1: AGGCCT-CTGCTTGCCGTTTGTTTCG and Tgnos2: CGCCGGCGGCACACGCTCAACTAATTAC including the restriction sites Stu1 and Not1 (underlined in the primer sequences) were used to amplify the Toxoplasma NOS gene by RT-PCR assay as described previously (Gutierrez-Escobar and Gómez-Marin, 2005) and then the product was recovered from agarose. The transfer vector pAcHLTA was digested with Stu1 and Not1 restriction enzymes. The purified Toxoplasma NOS PCR product amplification was ligated into the Stu1 and Not1 sites of the baculovirus transfer vector pAcHLT-A (Becton–Dickinson, Franklin Lakes, NJ, USA). The resulting plasmid was designated pAcHLT-A-NOS.
NO synthesis by Toxoplasma NOS recombinant protein of T. gondii was measured in a microplate assay for nitrite based on the Griess reaction (Stuehr and Marletta, 1985) and using the Nitric Oxide Synthase Assay Kit (Bioxytech, Foster City, USA) as recommended by the manufacturer. The reaction was run at room temperature for 40 min. Residual NADPH, which could interfere with the colorimetric assay, was removed at the end of the incubation by adding 40 U/ml of lactate dehydrogenase. The stability of the protein was determined by incubating the protein for 10 min at a range of temperatures between 0 and 100 °C and then the NOS assay was performed. The pH range for the recombinant NOS was evaluated using sodium acetate buffer at pH 4.5, potassium phosphate buffer at pH 7.0 and 7.5, and Tris–HCl buffer at pH 9.5 in the colorimetric assay.
2.4. Construction of recombinant baculovirus and DNA sequencing 2.8. Statistics Sf9 cells were cotransfected with the recombinant transfer vector pAcHLT-A-NOS and linear AcNPV viral DNA (Becton–Dickinson, Franklin Lakes, NJ, USA) following the manufacturer instructions for the Baculovirus Expression and Purification Kit (Clontech, California, USA). After 4 days incubation at 27 °C, the cells and supernatant containing the recombinant virus were harvested and a recombinant baculovirus was obtained. A recombinant transfer vector pAcHLT-XyIE was used as a control virus. Sequencing of recombinant plasmids was performed from Toxoplasma NOS PCR products. Specific bands were cut from low-melt agarose gels, followed by recovery using the Wizard PCR Prep kit (Promega, WI). Sequencing was done by using the ABIPRISM model 3100 version 3.7 at the Wadsworth Genetics Institute facility on purified DNA by using 2 pmol of the Tgnos1 and Tgnos primers. 2.5. Recombinant Toxoplasma NOS protein purification and electrophoretic analysis The sf9 cells were treated with lysis buffer and the supernatant was cleared by centrifugation. Later, they were added to a vial with prewashed Ni–NTA (Ni2 + nitrilotriacetate) beads (Clontech, CA, USA). The mixture was incubated at 4 °C for 1 h. The beads were collected, washed with lysis buffer and then, the Toxoplasma NOS protein that was bound to the beads, was eluted with lysis buffer containing 250 mM imidazole according to the manufacturer instructions. Afterwards, the protein eluted was quantified by the Bradford method. Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (trycine SDS–PAGE) was performed as described by Schägger (2006).
All data are expressed as means ± SEM from three experiments for NOS activity and in duplicate for kinetic, temperature and pH experiments. 3. Results 3.1. Cloning and bioinformatics analysis of Toxoplasma NOS-like gene The product of reverse transcription was amplified revealing a band with the expected size (237 pb) for a Toxoplasma NOS-like gene (Fig. 1). No amplification product was observed in the negative control, indicating that there was no contamination with genomic DNA. The similar sizes of bands obtained both in PCR and in RT-PCR reactions indicated that Toxoplasma NOS-like gene consisted of a single exon and this was further confirmed sequencing the cloning amplification products. During cloning, we used primers with restriction sites obtaining similar bands (Fig. 1). The Toxoplasma NOS product was purified from RT-PCR (Fig. 1, Lane 3) and then ligated into the baculovirus transfer vector pAcHLT-A and cloned into Escherichia coli DH5a strain. The recombinant vector was used in a Toxoplasma NOS gene PCR assay (Gutierrez-Escobar and Gómez-Marin, 2005) and sequenced (Fig. 2). The analysis of the sequence by using basic local alignment tool (BLAST) in ToxoDB (http://toxoDB.org) showed 100% of identity with our previously reported Toxoplasma NOS gene listed in the ToxoDB database (Fig. 2B). Furthermore, the Toxoplasma NOS gene was not present in the genome of the Sf9 culture cells after
2.6. Animal antisera and fluorescence staining One New Zealand White rabbit (2 kg) was housed, fed and handled in accordance with the recommendations made by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA). One hundred fifty micrograms of purified NOS protein emulsified in hydroxide aluminum adjuvant was inoculated intramuscularly at four sites. The same amount of immunogen mixed with aluminum hydroxide was inoculated as a booster on
Fig. 1. Toxoplasma NOS gene RT-PCR amplification in Toxoplasma gondii. Lane 1, positive kit control; lane 2, RT-PCR of DNase-treated total T. gondii RNA using the primers without restriction sites; lane 3, the RT-PCR of DNase-treated total T. gondii RNA using the primers with restriction sites; lane 4, negative control.
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Fig. 2. PCR assay of Toxoplasma NOS gene from pAcHLT-NOS. (A) Lanes 1 and 2, PCR products from the recombinant transfer vector pAcHLT-A-NOS by using primers with restriction sites; lane 3, PCR product from recombinant transfer vector pAcHLT-A-NOS by using primers without restriction sites; lane 4, negative control. (B) BLAST analysis at ToxoDB database of the sequence obtained from the Toxoplasma NOS PCR product (lane 3). (C) Clustal multiple alignment of the oxygenase domains of Drosophila melanogaster (NP_5233541), Deinococcus radiodurans (Q9RR97) and Bacillus halodurans (NP_241475) with the Toxoplasma NOS-like recombinant deduced aminoacid sequence.
the cotransfection process (Fig. 3). The recombinant NOS gene sequence was deposited in the GenBank (Accession No. EF452680). The deduced aminoacid sequence (R P L L A V C F V S D F Y S L S LLHFASVPFHESDGCVGRSHWLPGKHANYVKP A G A R K R P E V G C R S S C L L R S V C C D I L S P V R T R G N Stop L S V C) showed that the NOS signature motif was conserved (GCVGRS). The deduced aminoacid sequence was searched against the ORF database at ToxoDB.org (consulted February 9, 2008) and matched with a sequence (ORF 1b-4) at the chromosome 1b between pairs bases: 1,352,191–1,352,429. A promoter region is found at a distance of 2373 bp in the region between nucleotides 1,354,800 and 1,356,600 (Mathieu Gissot, personal communication). No similar sequences were found in PlasmoDB with this ORF. Interproscan analysis at www.expasy.org looking for protein domains of the ORF sequence indicates a signal peptide region, between amino acids 1–28, and the NOS signature motif, between amino acids 29–35. Additionally, the recombinant sequence was aligned against the ORF sequence of ToxoDB and 79 of the 81 residues were identical. Only the first two residues (R and P) of the recombinant protein are not present at the ORF at ToxoDB database
(shown in bold); this amino acids come from the restriction sites artificially introduced for cloning purposes into the baculovirus. An important characteristic of the NOS-like coding sequence is the presence of an alternative codon (CTG, leucine) at the beginning of the open reading frame. The theoretical molecular weight for the recombinant sequence was estimated to be 8.9 kDa and to have a pI of 9.3. 3.2. Biochemical and enzymatic analysis of recombinant Toxoplasma NOS-like protein We obtained three fractions of Ni–NTA-purified Toxoplasma NOS-like protein, with a concentration of 339, 203 and 455 lg of protein by ml. The activity of the purified recombinant protein in the NOS colorimetric assay in each of the fractions was 21.8 ± 0.9; 5.2 ± 0.2 and 3.7 ± 1.01 nmol/min/ml per lg of protein, respectively. Subsequent experiments were performed with the fraction with the highest activity. Activity was reduced only by 33% in absence of NADPH and by 66% when cofactors were absent and it was nearly inhibited when L-arginine was absent (Table 1).
Fig. 3. Sf9 culture cell. (A) Healthy Sf9 cells cultured in TNM-FH medium before cotransfection process. (B) Toxoplasma NOS PCR control in genomic DNA of Sf9 cell. 1, Positive control (Toxoplasma gondii DNA); 2, Toxoplasma NOS PCR in genomic DNA of Sf9 cells; 3, negative control.
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Tricine–SDS–PAGE with the fraction with the highest production of nitrites showed an unique band with a molecular weight of 11.6 kDa, as estimated by calculating the relative electrophoretic mobility (Fig. 4). Kinetic analysis of the purified enzyme was made using a range of L-arginine concentrations from 0.5 to 40 mM. The kM for the recombinant protein, as estimated by the double reciprocal plot of kinetic analysis, was 2.3 mM L-arginine (Fig. 5). The temperature for a maximum enzymatic activity was measured by
Table 1 Nitric oxide synthase activity based on Griess colorimetric reaction Nitrites production per microgram of protein (nmol/ml/min) Condition Condition Condition Condition Control
A B C D
21 ± 1.07 7 ± 1.4 14.6 ± 0.97 0.7 ± 0.9 0±0
Data are means ± SEM. (A) Purified recombinant Toxoplasma NOS-like protein + Larginine + NADPH + Cofactors (BH4 + FAD + FMN + Hem + Calmodulin) + Nitrite reductase. (B) Without Cofactors (BH4 + FAD + FMN + Hem + Calmodulin). (C) Without NADPH. (D) Without L-arginine. Control: Distilled water with L-arginine + Cofactors + Nitrate reductase.
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incubating the protein during 10 min at 0, 25, 37, 60 and 100 °C. The enzyme showed a maximum activity at 25 °C and lost about 60% of its activity when it was incubated at 37 °C (Fig. 6a). The optimum pH of the recombinant NOS occurred between 7.0 and 7.5 using potassium phosphate buffer (Fig. 6b). Activity was maximal after 40 min of incubation of enzyme and substrate (Fig. 6c). 3.3. Immunodetection of NOS-like protein The polyclonal antibody was first tested in an ELISA by using the His- tagged recombinant proteins for recombinant Toxoplasma NOS-like, metalloprotease or Entamoeba histolytica cysteine protease as an antigens coated to the plate wells. The absorbance of the preimmune serum was 0.06 ± 0.00 and after 4 weeks of inoculation the absorbencies for recombinant NOS-like was 0.44 ± 0.05, for recombinant Toxoplasma metalloprotease, 0.08 ± 0.0 and for recombinant Entamoeba histolytica cysteine protease, 0.08 ± 0.0. The serum obtained after 4 weeks of post-immunization was used in an immunofluorescence assay at dilutions of 1/50 and 1/10. A specific diffuse labeling of the entire body of the tachyzoites was observed (Fig. 7). 4. Discussion
Fig. 4. Tricine–SDS–PAGE of the Toxoplasma NOS recombinant protein. Arrows signals the bands for the Toxoplasma NOS recombinant protein and the bovine lactoalbumin molecular weight control marker (14 kDa).
Previously we identified the Toxoplasma NOS putative gene sequence and determined that the NOS sequence in Toxoplasma is a primitive one, as could be inferred from the phylogenetic analysis with the NOS sequences currently known (Gutierrez-Escobar and Gómez-Marin, 2005). Here, we have performed an initial preliminary biochemical characterization of the recombinant Toxoplasma NOS-like protein obtained from this genomic sequence. The enzymatic assays showed that the recombinant protein is able to produce nitrites from L-arginine. Antibodies against this recombinant protein specifically labeled Toxoplasma tachyzoites, confirming that it is a protein present in the parasite. Nitric oxide synthase enzymes in mammals are composed of oxygenase and reductase domains. However, bacterial NOS homologs, in the same manner as the Toxoplasma NOS-like pro-
Fig. 5. Kinetic analysis of the recombinant Toxoplasma nitric oxide synthase-like protein.
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Fig. 6. Nitrites production of the recombinant Toxoplasma nitric oxide synthase-like protein: (a) Effect of temperature on stability of the enzymatic activity of the protein. (b) Effect of modifications on pH buffer during nitrites assay. (c) Effect of different incubation time of the protein and substrate for the colorimetric assay.
Fig. 7. Immunofluorescence assay on tachyzoites with rabbit polyclonal antibodies anti-recombinant Toxoplasma NOS-like protein: (a) Control with preimmune rabbit serum 1/10. (b) 4 week post-immune rabbit serum at a dilution 1/50. (c) 4 week post-immune rabbit serum at a dilution 1/10.
tein, do not have the reductase domain. Thus, the critical question is how electrons are being donated to the oxygenase domain. In a structure modeling for bacterial NOS without reductase domains, Zemojtel et al. (2003) proposed that the heme-binding loops as well as the heme groups fit well in the cleft present in the vicinity of the NOS oxygenase N-terminal region. This model is therefore a possible configuration of a docking mode for bacterial NOS oxygenase domain which would not be possible for eukaryotic NOSs with an extended N-terminal part. This could explain how the oxygenase domain catches electrons without a reductase domain. Our kinetic measurements show that Toxoplasma NOSlike protein is not as efficient as mammalian NOS; therefore the presence of the two domains being expressed in one amino acid chain should allow for a more efficient NO catalysis in mammals. Alternatively, in vivo, Toxoplasma NOS-like protein could interact with the product of other genes with reductase like functions that would increase the NOS activity, as it has been proposed for bacteria (Zemoj-
tel et al., 2003). Some prokaryote NOS proteins, such as those from Deinococus, bound arginine and cofactor H4B, and had a normal heme environment. This despite missing N-terminal structures that bind Zn, and the dihydroxypropyl side chain of H4B, which help form an active dimer in mammalian NOS (Adak et al., 2002). Additionally the size of the recombinant Toxoplasma NOS-like protein is very small (11.6 kDa), similar to the 26 kDa NOS protein reported in E. histolytica (Hernandez-Campos et al., 2003) but smaller than the 40 kDa prokaryotic NOS protein in Bacillus subtilis (Adak et al., 2002) and that of 52 kDa in Nocardia species (Chen and Rosazza, 1995). The NOS gene sequence in Entamoeba is still unknown. The NOS proteins in mammals have been reported to be about 130 kDa (Moncada et al., 1991) and to be 160 kDa in arthropods such as Drosophila (Müller, 1994). The differences in the molecular weights in the NOS family therefore show an evolutionary divergence. Nitrite production by Toxoplasma NOS recombinant protein was approximately 21 nmol/min per lg of protein, more higher than that
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reported for E. histolytica NOS activity (5 nmol/min per lg of protein) (Hernandez-Campos et al., 2003) Our bioinformatic and biochemical results, suggest that T. gondii expresses a protein which is different from NOS isoforms found in mammalian cells and invertebrates but very similar to Deinococcus, and other bacterial NOS, which only contained an oxygenase domain. Previous observations indicated that T. gondii is an obligatory auxotroph for cytoplasmatic host cell L-arginine, because it does not have the enzymatic machinery for its synthesis (Fox et al., 2004). Furthermore, parasite cultures without L-arginine show an increase in the tachyzoites to bradyzoites interconversion (Fox et al., 2004). The present report is a first approach to analyze the characteristics of the recombinant Toxoplasma NOS-like protein and future work will study the localization and trafficking of the protein and characterize its catalytic parameters in both multiple and single turnover reactions. We also intend to study of inhibitors and analogs of L-arginine and address the role of calcium and others cofactors for Toxoplasma NOS activity. NO synthesis by a Toxoplasma NOS-like protein is now established, but a number of related issues remain to be explored. These include the crystal structure, the native redox partner(s), what Noxide product is generated during steady state synthesis, what conditions induce expression of these proteins, and whether allosteric factors exist that modulate kinetic parameters of the enzyme. Further research should therefore provide a deeper understanding of NOS structure/function and the evolutionary consequence of the NOS gene and its potential as chemotherapeutic target. Acknowledgments This work was supported by Colciencias, Grant No. 1113-0418247. We thank to Drs. Vern Carruthers and My-Hang Huynh from the Department of Microbiology of the University of Michigan for providing material, technical advice and critical discussion of the results and to Dr. Carlos Saavedra and Jason Isabelle, from the Wadsworth Institute, NY, for helping in sequencing recombinant DNA plasmids. References Adak, S., Aulak, K.S., Stuehr, J., 2002. Direct evidence for nitric oxide production by a nitric-oxide synthase protein from Bacillus subtilis. The Journal of Biological Chemistry 277, 16167–16171.
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