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Toxoplasma gondii: Identification of a putative nitric oxide synthase motif DNA sequence
Experimental Parasitology 111 (2005) 211–218 www.elsevier.com/locate/yexpr
Toxoplasma gondii: IdentiWcation of a putative nitric oxide synthase motif DNA sequence Andrés J. Gutierrez Escobar a, Jorge E. Gómez-Marin a,¤ a
Grupo de Estudio en Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindio, Armenia, Colombia Received 9 February 2005; received in revised form 10 August 2005; accepted 10 August 2005 Available online 26 September 2005
Abstract Nitric oxide (NO) plays an important role as a mediator of the immune response to intracellular pathogens in mice; however discordant results were obtained in in vitro human cell lines experiments. Thus, we found that nitrite levels (nitric oxide derivatives) were increased in presence of Toxoplasma but not with IFN plus LPS treatment during Toxoplasma infection of a human monocyte cell line THP1 and Griess assays conWrmed that Toxoplasma alone has a nitrite production that was surprisingly increased by the most common inhibitor of nitric oxide synthase (NOS) in mammals. To look for genomic sequences that code for NOS gene in Toxoplasma, which could explain this production of NO derivatives, speciWc NOS motifs were sougth by bioinformatics methods. A putative NOS motif sequence was found in one contig of the Toxoplasma genome (TGG_994574). SpeciWc primers ampliWed a segment of 270 bp in RT-PCR assay, indicating that it is a transcription gene in the tachyzoite stage. Our results are the Wrst description of the existence and transcription of a putative NOS DNA sequence in a pathogenic protozoan. 2005 Elsevier Inc. All rights reserved. Keywords: Toxoplasma; Nitric oxide synthase; Griess assays; Protozoa; Bioinformatics
1. Introduction As a consequence of studies in animal models, the nitric oxide (NO) molecule has been designated as a principal mediator of the immune response to intracellular pathogens; however, discordant results were obtained in in vitro human cell lines experiments (Murray et al., 1985). We studied the eVector mechanisms of interferon gamma against infection by Toxoplasma gondii in a human monocytic cell line and we found that nitric oxide was not involved as a protector mechanism and that T. gondii has an innate production of nitric oxide derivatives (Gomez and Marin, 2000). This suggests that T. gondii possesses a nitric oxide synthase (NOS) capability. Genes coding for NOS have been found present throughout the plant and
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Corresponding author. Fax: +5767460129. E-mail address: [email protected] (J.E. Gómez-Marin).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.08.004
animal kingdom during the last years (Korneev and O’Shea, 2002). Their primary structures and activities are strikingly similar to mammalian NOS, suggesting that NO has been important throughout evolution. All eukaryotic NOS catalyze the NADPH- and O2-dependent oxidation of L-arginine (Arg) to citrulline and NO. Structurally, all animal NOS are bi-domain proteins containing an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H4B), and Arg and is linked to a C-terminal Xavoprotein domain (NOS reductase domain, NOSred) by a central calmodulin (CaM) binding sequence. NOSred bears strong sequence and functional similarity to NADPH-cytochrome P450 reductase and related electron transfer Xavoproteins, and function to transfer NADPH-derived electrons to the ferric heme for O2 activation during NO synthesis (Alderton et al., 2001). In contrast, NOSoxy and cytochromes P450s have completely diVerent primary, secondary, and tertiary structures, even though both enzyme families use a thiolate-ligated
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heme for O2 activation. Moreover, unlike P450s, NOSoxy must dimerize to become active (Alderton et al., 2001). Dimerization produces functional binding sites for Arg and H4B and sequesters the heme catalytic center from solvent. These distinguishing features imply that NOS evolved separately from other heme-thiolate enzymes and can provide unique perspectives on their structure- function relationships and that NO is one of the earliest and most widespread signaling molecules in living organisms. In this article, we describe the bioinformatic search and the experiments performed to demonstrate the existence and transcription of a putative NOS coding DNA sequence in the human opportunistic pathogen protozoan T. gondii. 2. Materials and methods 2.1. Cell culture and parasites (live parasites or total antigen and secretory/excretory fractions) The human myelomonocytic cell line (THP1) (202 TIB, American Type Cellular Collection, Rockville, MD) was maintained in RPMI 1640 medium containing 10% heatdecomplemented FBS, 2 M glutamine, 1000 U/ml penicillin, and 100 g/ml streptomycin at 37 °C in a humidiWed atmosphere containing 5% CO2. The cell density was 2–5 £ 105 cells/ml in 50–75 ml total volume. Cells were harvested twice weekly in 25 cm2 cell culture Xasks. Female Swiss Webster mice were inoculated with T. gondii RH strain and tachyzoites were recovered from the peritoneal cavity 3 to 4 days later by instilling 5 ml sterile 0.9% NaCl solution with antibiotics (penicillin 100 U/ml and clamoxicillin 100 g/ml). Tachyzoites were isolated by centrifugation twice at 200g for 10 min. The pellets were resuspended in RPMI medium and Wltered through 3 m 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 cells or parasites were used. For total antigen lysate, tachyzoites were resuspended in saline solution and submitted to freeze–thawing four times and then disrupted by sonication (4 £ 20 W for 20 s) using a microprobe. After centrifugation at 5000 rpm £ 20 min, supernatant was recovered and used for nitrite determination. For secretory/excretory antigen 1.5 £ 108 tachyzoites were Wltered through 3 m Wlter and resuspended on invasion milieu (DMEM, 20 mM Hepes, fetal calf serum 3%). Parasites were induced to secretion by A23187 0.4 M and supernatant recovered after centrifugation 10 min at 1000g and Wltered through 0.22 m membrane (Miller et al., 2001). Protein amounts were dosed by the method of Lowry.
and experiments repeated at least three times. In vitro infected culture were maintained at 37 °C in a humidiWed atmosphere containing 5% CO2 and supernatants were harvested at 6, 24, and 48 h after infection. 2.3. Determination of nitrite To test the production of nitrite as a stable end product of NO synthesis, supernatant of a culture of THP1, a human monocytic cell line, were harvested from wells either infected or not with T. gondii and treated or not treated with IFN 1000 U/ml combined with Escherichia coli puriWed lypolysacharide (LPS) 10 g/ml. Nitrite production was also tested in noninfected cell cultures and on supernatants of T. gondii without human cells. Cell density in noninfected cells was the same used for infected cultures (2 £ 105 cells/ml). Toxoplasma without human cells was maintained in saline solution in wells with increasing number of parasites (1–18 £ 106 tachyzoites). Nitrite production was determined by a microplate assay method (Grisham et al., 1999). BrieXy, 100 l samples were removed from the supernatants and incubated with equal volume of Griess reagent (1% sulfanilamide/0.1% N-1 napthylethylene diamine dihydrochloride/2.5% H3PO4) at room temperature for 10 min. The absorbance at 550 nm, using a reference Wlter of 630 nm, was determined with a microplate reader. Nitrite concentration was calculated using a standard solution of sodium nitrite (NaNO2) 0.05–500 m. 2.4. Inhibition of NO synthesis and analysis of T. gondii growth in THP1 cultures To test the role of NO in overall growth of cell cultures infected with T. gondii, cell cultures were incubated for 48 h in medium alone (control) or in medium containing human recombinant IFN (1000 U/ml)—Boehringer Ingelheim, Germany- plus LPS (10 mg/ml)—Sigma, USA- in the absence or presence of 1 M NG monomethyl-L-arginine (NGMMA, Sigma, USA). The growth of tachyzoites in THP1 cell culture was evaluated by counting the total number of tachyzoites per 100 infected and not infected randomly selected cells (an assay of overall T. gondii growth in THP1 cell culture). The cells were stained using the RAL staining kit (RAL reactifs S.A., Paris, France). At least 300 cells from each well were counted. The experiments were then carried out in triplicate for each indicated condition and repeated at least three times. 2.5. Sequence data and bioinformatics analysis tools
2.2. In vitro infection THP1 cells were harvested at the exponential growth stage (day 3) and cultured in 24 well plates (2 £ 105 cells per well). Approximately 2 £ 105 parasites were added in each well in 1 ml of Wnal volume (parasite cell ratio 1:1). Three wells were assayed for each investigated condition
Sources and/or Accession Numbers for NOS and related sequences were as follows: NOS3 (gi266648), iNOS (gi1352513), and nNOS (gi1709333). DNA and protein sequences were analyzed for homologues using BLAST programs at http://toxodb.org/restricted/toxoDBLAST.shtml (Kissinger et al., 2003). Protein mapping and motif searches
A.J. Gutierrez Escobar, J.E. Gómez-Marin / Experimental Parasitology 111 (2005) 211–218
were performed at WU-BLAST, Pfam (http://pfam. wustl.edu) and Prosite (www.expasy.org). Multiple sequence alignments were performed using ClustalW at http://www.ebi.ac.uk/clustalw/. Phylogenetic analysis was performed with software Evolutionary trace at http:// www-cryst.bioc.cam.ac.uk/jiye/evoltrace/evoltrace.html and Mega (www.megasoftware.net). Analysis of coding DNA sequence (CDS) on the Toxoplasma genome contig was performed with Artemis release 6.0 (www.sanger.ac.uk). 2.6. PCR for genomic DNA Tachyzoites isolated from infected mice were used for T. gondii RH strain DNA extraction that was performed using the Wizard DNA puriWcation kit (Promega, Madison, Wis.) in accordance with the manufacturer’s instructions. We used DNA from representative strains of the three lineages of Toxoplasma for complementary experiments: Martin strain (type I), SQM strain (type II), and CEP strain (type III), kindly donated by Dr. Darde (Limoges, France). AmpliWcation of NOS motif was performed using a PCR mix consisted of 75 mM Tris–HCl (pH 9.0), 50 mM KCl, 20 mM (NH4)2SO4, and 2 mM MgCl2, 0.001% bovine serum albumin, 200 M each of the two deoxynucleoside triphosphates, 1.8 M of each primer, 1.5 U of DNA polymerase (PCR Super Mix, Promega), and, as a template, 1 l of extracted sample DNA in a Wnal volume of 25 l. All the PCRs were performed in an Amplitron II thermal cycler (Thermolyne, USA). The Wrst step of ampliWcation was 30 s of denaturation at 94 °C. This step was followed by 40 cycles, with 1 cycle consisting of 30 s at 94 °C, 30 s at the 58 °C annealing temperature for each primer, and 30 s at 72 °C. The Wnal cycle was followed by an extension step of 10 min at 72°C. The sequences of primers were: Tgnos1: CTGCTTGCCGTTTGTTTCG and TgNOS2: GCACACG CTCAACTAATTAC. The PCR products were analyzed by 2% agarose gel electrophoresis. To avoid possible contamination, several measures, such as separate space to set up PCRs and disposable Wlter tips, were taken, as well a negative control (no DNA), and positive controls from diVerent strains of T. gondii were used. 2.7. RT-PCR for transcription analysis For RT-PCR assays, total RNA was puriWed from Toxoplasma tachyzoites using TRIzol (Invitrogen, USA) reagent. Sterile tubes, pipette tips, gloves, and diethyl pyrocarbonate treated water was used in extraction procedures. First-strand cDNA from RNA was synthesized by using Avian Myeloblastosis Virus (AMV) reverse transcriptase from Access RT-PCR system (Promega, Madison, WI). The reaction was performed in a 45 l mixture containing a standard enzyme buVer supplied by the manufacturer: 10 l of RNA sample, downstream (Tgnos2 GCACACGCT CAACTAATTAC) and upstream primers (Tgnos1 CTGC TTGCCGTTTGTTTCG) at 1.8 M concentration/0.2 mM each dNTPs and 0.1 U/l of the enzyme. The reaction mix-
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ture was incubated at 48 °C for 45 min and then at 94 °C for 2 min to inactive AMV enzyme. 5 l of the mixture was used for PCR analysis as a template cDNA. The PCR ampliWcation was performed in 100 l reaction mixtures containing 1 £ PCR buVer supplied by the manufacturer (PCR Master Mix, Promega, USA), 2.5 mM MgCl2, 200 M dNTPs, 2.5 U of recombinant Taq DNA polymerase, each of the primers at 1.0 M, and template cDNA. The Wrst step of ampliWcation was 30 s of denaturation at 94 °C. This step was followed by 15–40 cycles, with 1 cycle consisting of 30 s at 94 °C, 30 s at the 58 °C annealing temperature for each primer, and 30 s at 72 °C. The Wnal cycle was followed by an extension step of 10 min at 72 °C. RTPCR products were runned on 2% agarose electrophoresis gels and then visualized in an ultraviolet lamp. As a control in cDNA template for a housekeeping gene in tachyzoite stages we use tubulin primers (sense: AGTCCAGCGTCTGTGACATCC; antisense: GCACCCATCTCGCCCTCTTCC) and we performed 40 PCR cycles with 1 cycle consisting of 30 s at 94 °C for denaturation, 1 min at 62 °C for annealing, and a Wnal extension step of 1 min at 72 °C. 3. Results 3.1. NO inhibitor signiWcantly increase T. gondii growth in THP1 cells and the nitrites production (NO derivatives) of Toxoplasma During experiments on eVector protective mechanisms of IFN plus bacterial LPS the overall growth of intracellular Toxoplasma was increased in the presence of 1 M of the NO inhibitor NG monomethyl-L-arginine (NGMMA). This was signiWcantly reduced by IFN + LPS (Fig. 1). To understand the enhanced growth of Toxoplasma in the presence of NGMMA, the levels of nitrite (a stable NO derivative) were measured in supernatants of THP1 cell in the presence or not of Toxoplasma and treated or untreated with IFN plus LPS culture. IFN with LPS on THP1 culture cells, without the parasite, did not increase NO derivatives levels (Fig. 2). In contrast, Toxoplasma infection of THP1 cells (Fig. 2) and the supernatants of Toxoplasma maintained alone in saline solution (Fig. 3) produced 2–4 M of nitrites. The levels in supernatants were similar at concentration of 1 £ 106 or at 18 £ 106 tachyzoites concentration but NGMMA addition increased the nitrite concentration of tachyzoites alone in a linear fashion (Fig. 3). In addition, fractions of total lysate antigen (TLA) prepared after sonication of T. gondii tachyzoites or of secretory/excretory antigen (E/ S) with or without NGMMA, did not shown any signiWcant levels of nitrites (absorbance in TLA was 0.0 without NGMMA and 0.007 with NGMMA and in E/S was 0.0 without NGMMA and 0.0011 with NGMMA, these absorbancy values were below the level of detection of the Griess assay).
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Fig. 1. Overall growth of Toxoplasma in THP1 cells after 48 h of culture, calculated as the number of intracellular parasites by 100 cells in THP1 cultures in the absence (control) and in the presence of IFN 1000 U/ml plus LPS 10 g/ml (IFN + LPS) or in the absence or presence of 1 M NGMMA (+NGMMA). SigniWcant diVerences were found between (*) Control vs. IFN + LPS (t test p D 0.015) and between (**) NGMMA vs IFN + LPS + NGMMA (t test p D 0.000). The number of tachyzoites was determined by counting cells stained with a modiWed Giemsa stain. Bars represent means § SD of triplicates obtained in one experiment; similar results were obtained in three separate experiments.
Fig. 2. Nitrite levels in THP1 infected and treated or not with IFN plus LPS measured in THP1 cell culture supernatant by Griess reagent after 24 h of culture. Bars represent means § SE of triplicates (n D 9) of three separate experiments. Paired t test demonstrated statistically signiWcant diVerences in NO2 levels between Cell vs. Cell + T. gondii (Mann–Whitney nonparametric test p D 0.015). NaNO2 standard curve was from 0.05 to 500 M.
Fig. 3. Nitrite levels from Toxoplasma tachyzoites maintained alone in the presence or not of NGMMA and measured on saline solution supernatants after 24 h of culture. Nitrites levels were obtained by using Griess reagent and levels calculated by using a NaNO2 (Sigma) standard curve 0.05–500 M.
LLAVCFVSDFYSLSLLHFASVPFHESDGCVGRSH WLPGKHANYVKPAGARKRPEVGCRSSCLLRSVCC DILSPVRTRGNLSVCKYALVLSIACRGRSLLAGIQA LFEMPDLVTALIATMLFSGVLVIWNAWMCAAVLT IGRPVPKPLTSDAGEILGVETDFLAPAWRPVALGV VILSHLLQTETS. The translated aminoacid sequences have a predicted molecular weight of 20 kDa and include: a peptide signal, a transmembrane region, a glycosylation site, a N-myristoylation site and a NOS signature motif (Fig. 4). Multiple alignments of DNA sequences were performed with eNOS, iNOS, and nNOs sequences. Only the critical aminoacids are conserved in the NOS (C, G, and R residues) and in myristyl (V and C residues) motifs (Fig. 5). In NOS signature motif four residues are conserved along the phylogeny: C, G, R, and W. A phylogenetic tree placed Lymnea NOS motif sequence as the nearest sequence to the Toxoplasma NOS motif. Lymnea, Toxoplasma, and bacterial NOS are part of the same divergent
3.2. IdentiWcation of a NOS motif sequence in the Toxoplasma genome One deduced aminoacid genomic sequence of the contig TGG_994574 of ToxoDB, contained the motif NOS speciWc sequence motif [GR]C[IV]GR[ILS]W. We found, by using the Artemis software, a complete ORF sequence (561 bp) that begins at 152,107 and ends at 152,565 bp of the contig:
Fig. 4. Diagram of the protein domains in the Toxoplasma NOS sequence as obtained after submitting sequence to Prosite. Abbreviations, SP, Signal peptide; NOS, NOS motif; N-Myr, N-Myrystoil; Gly, Glycosylation; Tm, transmembrane domain. The sequence was obtained between 152,107 and 152,565 bp of the contig TGG_994574 of ToxoDB (toxodb.org).
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Fig. 5. Clustal multiple alignment of NOS sequences. (A) Multiple alignment with NOS of diVerent animal species; Boxes: * NOS motif, ** Myrystoil motif. (B) Residues pattern indicating the phylogenetic NOS motif (CXGRXXW). The sequences used for alignment were: gi冷266648冷 sp冷P29474冷 NOS3_HUMAN nitric oxide synthase, (endothelial NOS or eNOS); gi冷1352513冷 sp冷P35228冷 NS2A_HUMAN Nitric oxide synthase (inducible NOS or iNOS); gi冷1709333冷 sp冷P29475冷 NOS1_HUMAN nitric oxide synthase, brain (neuronal NOS or nNOS).
ancestor molecule that divide early into a group that incorporated a reductase domain (which includes the mammalian and metazoan NOS) and those that did not (Fig. 6). By using speciWc primers for a segment of the ORF, the PCR on genomic DNA obtained a 237 bp product. The molecular weight of the product was as predicted by in silico analysis (Fig. 7A). This sequence was ampliWed in many diVerent strains of Toxoplasma indicating that is conserved in the type I, type II, and type III lineages of Toxoplasma (Fig. 7B). 3.3. Putative NOS motif transcription RT-PCR analysis was performed to demonstrate that NOS motif was a transcription sequence. No DNA contamination was observed in Toxoplasma tachyzoite puriWed RNA. We used 33 g of Toxoplasma RNA to obtain cDNA. After 35 cycles, we found a plateau of PCR ampliWcation assays with the cDNA and we obtained a species speciWc 237 bp product (Fig. 8). The size of the product was as expected by the in silico analysis.
4. Discussion For many years the defensive role of NOS in human macrophages was a controversial topic, but it is now generally accepted that this is not an important mechanism in human toxoplasmosis (Gomez and Marin, 2000). Thus, levels of nitrites during activation of inducible NOS in mouse can reach 120 M or more but only 3–6 M in supernatant of human monocytes. Our experiments demonstrate that Toxoplasma, like other protozoan such as Tetrahymena (Christensen, 1996), Trypanosoma (Paveto et al., 1995), and Plasmodium (Ghigo et al., 1995), can have its own nitrite production that would reXect constitutive NOS activity (producing 2–6 M of nitrites). Genestra et al. (2003) have also shown that Leishmania produce levels of nitrite similar to those we found in Toxoplasma; however in their case, there was inhibition by several L-arginine analogs. We did not expected replication of Toxoplasma tachyzoites in medium without host cells. The experiment was performed over 24 h, which was long enough to keep Toxoplasma alive (Gómez Marin et al., 1996), and obtain nitrite production
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Fig. 6. Phylogenetic tree obtained using Mega software. Statistical method was the neighborhood joining tree and using 1000 bootstrapping replications.
by the parasite without nitrite production by host cells. We also measured levels of nitrites in parasite fractions (total antigen and excretory/secretory antigen) and since nitrites production was not found, indicating that NOS enzyme was not functional after this procedure of fractioning. We think that NOS activity in T. gondii was presumably induced (but not inhibited) by NGMMA, in live tachyzoites. Previous work has showed that NOS activity in THP1 cells is not a protective mechanism induced by IFN against Toxoplasma, and enhanced growth of T. gondii in the presence of NGMMA cannot be explained by an eVect on host NOS (Gomez and Marin, 2000). Nitrite production could be the result of many nitrogen consuming pathways (e.g., by the arginine deaminase enzyme activity). However, we consider, in the case of Toxoplasma, that nitrites production could also be the result of the NOS pathway. The existence of a putative NOS motif signature DNA sequence that is transcribed is a very strong argument to believe that nitrites in Toxoplasma would be derived from a NOS enzyme. The NOS motif [GR]C[IV]GR[ILS]W sequence that was used for the in silico search is the site where L-arginine bind to the eNOS enzyme. The sequence found in Toxoplasma was 561 bp long and has a low predicted molecular weight. The other regions predicted by the translated sequence, such as the N-Myrystoil site are important for the posttransductional membrane translation of the protein. The
membrane localization of the Toxoplasma NOS coded protein is also supported by the existence of the transmembrane domain. The presence of these motifs in the NOS sequence of Toxoplasma suggests that it is more related functionally to endothelial NOS (Raman et al., 1998). The NOS sequence in Toxoplasma is a primitive one as could be inferred by the phylogenetic analysis with the NOS sequences that are known currently. The NOS proteins included in the phylogenetic analysis showed silent mutations in the NOS motif such as a change of isoleucine to valine or to leucine (hidrophobes residues). Other residues changes such as the hydrophylic glutamine replaced by tirosine residues in mammals would be not as important as a change of glutamine to phenylalanine in bacterial NOS. The NOS conserved residues (C, G, R, and W) are evolutionary traces and deWnitive markers (phylogenetic motif) for the NOS protein family (Korneev and O’Shea, 2002). The phylogenetic tree suggests that nNOS and eNOS are the product of more ancillary gene duplication than those occurred for iNOS. Bacterial NOS is an independent branch that originated from this early duplication. One remarkable characteristic in mammals NOS is the presence of reductase domains that are absent in the Toxoplasma NOS and in bacterial NOS (Crane et al., 1998). One surprising Wnding was that one of best known NOS inhibitor increased the nitrites production by Toxoplasma.
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with the glutamate residue that binds to the L-arginine substrate (Crane et al., 1997). Kinetic studies with the puriWed NOS of Toxoplasma protein would be required to determine an allosteric eVect of NGMMA. Previously, NOS enzyme has been isolated from other protozoa; Basu et al. (1997) puriWed a NOS protein from Leishmania donovani. Present results encourage us to continue exploring the Toxoplasma NOS sequence as a probable therapeutic target; Wrst, the RT-PCR assays indicate that the putative NOS sequence is a transcriptionally active one. Second, NGMMA signiWcantly increases the growth of Toxoplasma in cell culture indicating that NOS it would have an important role stimulating parasite metabolism. As NGMMA has the opposite eVect on mammalian NOS, this suggests that Toxoplasma NOS has a unique structure that could permit the design of speciWc inhibitors without interference with mammalian NOS. In conclusion, we have identiWed the Wrst putative NOS coding DNA sequence in a protozoan pathogen; further studies are warranted to purify and to analyze its biochemical and functional characteristics. Fig. 7. Putative NOS PCR assay, genomic DNA was obtained from the tachyzoite stage of Toxoplasma. (A) MW: Molecular weight marker Puc18 digested with BamH1. 1:Escherichia coli DH5 strain. 2: Taenia solium. 3: Homo sapiens leukocytes. 4: T. gondii Rh strain. 5: Negative control (PCR mix without DNA). DNA concentrations of samples were checked by electrophoresis before PCR. (B) 1: Negative control (PCR mix without DNA). 2: DNA from Martin Toxoplasma strain (type I lineage), 3: SQM Toxoplasma strain (type II lineage), and 4: CEP Toxoplasma strain (type III lineage).
Acknowledgments Andres Gutierrez was beneWciary of a grant from the Young Researcher Program from Colciencias (Colombia). We thank specially to Aylan Arenas and Jorge Jimenez who collaborate in some Wnal experiments. Preliminary genomic and/or cDNA sequence data was accessed via http://ToxoDB.org and/or http://www.tigr.org/tdb/t_gondii/. Genomic data were provided by The Institute for Genomic Research (supported by the NIH Grant #AI05093), and by the Sanger Center (Wellcome Trust). EST sequences were generated by Washington University (NIH Grant #1R01AI045806-01A1). We thank Dr. Herney Bolivar who checked our English spelling. References
Fig. 8. RT-PCR assay for putative NOS sequence. MW: Molecular weight marker Puc18 digested with BamH1. 1: Toxoplasma tubulin ampliWed by using the speciWc primers as described in Section 2. 2: SpeciWc ampliWcation of putative NOS sequence from cDNA of T. gondii RH strain tachyzoite. 3 and 4: RT negative control to rule out contaminating DNA was performed by using a RT reaction product without reverse transcriptase during cDNA synthesis.
NGMMA is L-arginine structural analog and inhibits NOS in mammals by competing with binding to the NOS motif of L-arginine. Crystallographic studies have found that NGMMA and other NOS speciWc inhibitors like aminoguanidine, S-ethylthiourea and thiocitruline directly interacts
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Toxoplasma gondii: IdentiWcation of a putative nitric oxide synthase motif DNA sequence Andrés J. Gutierrez Escobar a, Jorge E. Gómez-Marin a,¤ a
Grupo de Estudio en Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindio, Armenia, Colombia Received 9 February 2005; received in revised form 10 August 2005; accepted 10 August 2005 Available online 26 September 2005
Abstract Nitric oxide (NO) plays an important role as a mediator of the immune response to intracellular pathogens in mice; however discordant results were obtained in in vitro human cell lines experiments. Thus, we found that nitrite levels (nitric oxide derivatives) were increased in presence of Toxoplasma but not with IFN plus LPS treatment during Toxoplasma infection of a human monocyte cell line THP1 and Griess assays conWrmed that Toxoplasma alone has a nitrite production that was surprisingly increased by the most common inhibitor of nitric oxide synthase (NOS) in mammals. To look for genomic sequences that code for NOS gene in Toxoplasma, which could explain this production of NO derivatives, speciWc NOS motifs were sougth by bioinformatics methods. A putative NOS motif sequence was found in one contig of the Toxoplasma genome (TGG_994574). SpeciWc primers ampliWed a segment of 270 bp in RT-PCR assay, indicating that it is a transcription gene in the tachyzoite stage. Our results are the Wrst description of the existence and transcription of a putative NOS DNA sequence in a pathogenic protozoan. 2005 Elsevier Inc. All rights reserved. Keywords: Toxoplasma; Nitric oxide synthase; Griess assays; Protozoa; Bioinformatics
1. Introduction As a consequence of studies in animal models, the nitric oxide (NO) molecule has been designated as a principal mediator of the immune response to intracellular pathogens; however, discordant results were obtained in in vitro human cell lines experiments (Murray et al., 1985). We studied the eVector mechanisms of interferon gamma against infection by Toxoplasma gondii in a human monocytic cell line and we found that nitric oxide was not involved as a protector mechanism and that T. gondii has an innate production of nitric oxide derivatives (Gomez and Marin, 2000). This suggests that T. gondii possesses a nitric oxide synthase (NOS) capability. Genes coding for NOS have been found present throughout the plant and
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Corresponding author. Fax: +5767460129. E-mail address: [email protected] (J.E. Gómez-Marin).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.08.004
animal kingdom during the last years (Korneev and O’Shea, 2002). Their primary structures and activities are strikingly similar to mammalian NOS, suggesting that NO has been important throughout evolution. All eukaryotic NOS catalyze the NADPH- and O2-dependent oxidation of L-arginine (Arg) to citrulline and NO. Structurally, all animal NOS are bi-domain proteins containing an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H4B), and Arg and is linked to a C-terminal Xavoprotein domain (NOS reductase domain, NOSred) by a central calmodulin (CaM) binding sequence. NOSred bears strong sequence and functional similarity to NADPH-cytochrome P450 reductase and related electron transfer Xavoproteins, and function to transfer NADPH-derived electrons to the ferric heme for O2 activation during NO synthesis (Alderton et al., 2001). In contrast, NOSoxy and cytochromes P450s have completely diVerent primary, secondary, and tertiary structures, even though both enzyme families use a thiolate-ligated
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heme for O2 activation. Moreover, unlike P450s, NOSoxy must dimerize to become active (Alderton et al., 2001). Dimerization produces functional binding sites for Arg and H4B and sequesters the heme catalytic center from solvent. These distinguishing features imply that NOS evolved separately from other heme-thiolate enzymes and can provide unique perspectives on their structure- function relationships and that NO is one of the earliest and most widespread signaling molecules in living organisms. In this article, we describe the bioinformatic search and the experiments performed to demonstrate the existence and transcription of a putative NOS coding DNA sequence in the human opportunistic pathogen protozoan T. gondii. 2. Materials and methods 2.1. Cell culture and parasites (live parasites or total antigen and secretory/excretory fractions) The human myelomonocytic cell line (THP1) (202 TIB, American Type Cellular Collection, Rockville, MD) was maintained in RPMI 1640 medium containing 10% heatdecomplemented FBS, 2 M glutamine, 1000 U/ml penicillin, and 100 g/ml streptomycin at 37 °C in a humidiWed atmosphere containing 5% CO2. The cell density was 2–5 £ 105 cells/ml in 50–75 ml total volume. Cells were harvested twice weekly in 25 cm2 cell culture Xasks. Female Swiss Webster mice were inoculated with T. gondii RH strain and tachyzoites were recovered from the peritoneal cavity 3 to 4 days later by instilling 5 ml sterile 0.9% NaCl solution with antibiotics (penicillin 100 U/ml and clamoxicillin 100 g/ml). Tachyzoites were isolated by centrifugation twice at 200g for 10 min. The pellets were resuspended in RPMI medium and Wltered through 3 m 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 cells or parasites were used. For total antigen lysate, tachyzoites were resuspended in saline solution and submitted to freeze–thawing four times and then disrupted by sonication (4 £ 20 W for 20 s) using a microprobe. After centrifugation at 5000 rpm £ 20 min, supernatant was recovered and used for nitrite determination. For secretory/excretory antigen 1.5 £ 108 tachyzoites were Wltered through 3 m Wlter and resuspended on invasion milieu (DMEM, 20 mM Hepes, fetal calf serum 3%). Parasites were induced to secretion by A23187 0.4 M and supernatant recovered after centrifugation 10 min at 1000g and Wltered through 0.22 m membrane (Miller et al., 2001). Protein amounts were dosed by the method of Lowry.
and experiments repeated at least three times. In vitro infected culture were maintained at 37 °C in a humidiWed atmosphere containing 5% CO2 and supernatants were harvested at 6, 24, and 48 h after infection. 2.3. Determination of nitrite To test the production of nitrite as a stable end product of NO synthesis, supernatant of a culture of THP1, a human monocytic cell line, were harvested from wells either infected or not with T. gondii and treated or not treated with IFN 1000 U/ml combined with Escherichia coli puriWed lypolysacharide (LPS) 10 g/ml. Nitrite production was also tested in noninfected cell cultures and on supernatants of T. gondii without human cells. Cell density in noninfected cells was the same used for infected cultures (2 £ 105 cells/ml). Toxoplasma without human cells was maintained in saline solution in wells with increasing number of parasites (1–18 £ 106 tachyzoites). Nitrite production was determined by a microplate assay method (Grisham et al., 1999). BrieXy, 100 l samples were removed from the supernatants and incubated with equal volume of Griess reagent (1% sulfanilamide/0.1% N-1 napthylethylene diamine dihydrochloride/2.5% H3PO4) at room temperature for 10 min. The absorbance at 550 nm, using a reference Wlter of 630 nm, was determined with a microplate reader. Nitrite concentration was calculated using a standard solution of sodium nitrite (NaNO2) 0.05–500 m. 2.4. Inhibition of NO synthesis and analysis of T. gondii growth in THP1 cultures To test the role of NO in overall growth of cell cultures infected with T. gondii, cell cultures were incubated for 48 h in medium alone (control) or in medium containing human recombinant IFN (1000 U/ml)—Boehringer Ingelheim, Germany- plus LPS (10 mg/ml)—Sigma, USA- in the absence or presence of 1 M NG monomethyl-L-arginine (NGMMA, Sigma, USA). The growth of tachyzoites in THP1 cell culture was evaluated by counting the total number of tachyzoites per 100 infected and not infected randomly selected cells (an assay of overall T. gondii growth in THP1 cell culture). The cells were stained using the RAL staining kit (RAL reactifs S.A., Paris, France). At least 300 cells from each well were counted. The experiments were then carried out in triplicate for each indicated condition and repeated at least three times. 2.5. Sequence data and bioinformatics analysis tools
2.2. In vitro infection THP1 cells were harvested at the exponential growth stage (day 3) and cultured in 24 well plates (2 £ 105 cells per well). Approximately 2 £ 105 parasites were added in each well in 1 ml of Wnal volume (parasite cell ratio 1:1). Three wells were assayed for each investigated condition
Sources and/or Accession Numbers for NOS and related sequences were as follows: NOS3 (gi266648), iNOS (gi1352513), and nNOS (gi1709333). DNA and protein sequences were analyzed for homologues using BLAST programs at http://toxodb.org/restricted/toxoDBLAST.shtml (Kissinger et al., 2003). Protein mapping and motif searches
A.J. Gutierrez Escobar, J.E. Gómez-Marin / Experimental Parasitology 111 (2005) 211–218
were performed at WU-BLAST, Pfam (http://pfam. wustl.edu) and Prosite (www.expasy.org). Multiple sequence alignments were performed using ClustalW at http://www.ebi.ac.uk/clustalw/. Phylogenetic analysis was performed with software Evolutionary trace at http:// www-cryst.bioc.cam.ac.uk/jiye/evoltrace/evoltrace.html and Mega (www.megasoftware.net). Analysis of coding DNA sequence (CDS) on the Toxoplasma genome contig was performed with Artemis release 6.0 (www.sanger.ac.uk). 2.6. PCR for genomic DNA Tachyzoites isolated from infected mice were used for T. gondii RH strain DNA extraction that was performed using the Wizard DNA puriWcation kit (Promega, Madison, Wis.) in accordance with the manufacturer’s instructions. We used DNA from representative strains of the three lineages of Toxoplasma for complementary experiments: Martin strain (type I), SQM strain (type II), and CEP strain (type III), kindly donated by Dr. Darde (Limoges, France). AmpliWcation of NOS motif was performed using a PCR mix consisted of 75 mM Tris–HCl (pH 9.0), 50 mM KCl, 20 mM (NH4)2SO4, and 2 mM MgCl2, 0.001% bovine serum albumin, 200 M each of the two deoxynucleoside triphosphates, 1.8 M of each primer, 1.5 U of DNA polymerase (PCR Super Mix, Promega), and, as a template, 1 l of extracted sample DNA in a Wnal volume of 25 l. All the PCRs were performed in an Amplitron II thermal cycler (Thermolyne, USA). The Wrst step of ampliWcation was 30 s of denaturation at 94 °C. This step was followed by 40 cycles, with 1 cycle consisting of 30 s at 94 °C, 30 s at the 58 °C annealing temperature for each primer, and 30 s at 72 °C. The Wnal cycle was followed by an extension step of 10 min at 72°C. The sequences of primers were: Tgnos1: CTGCTTGCCGTTTGTTTCG and TgNOS2: GCACACG CTCAACTAATTAC. The PCR products were analyzed by 2% agarose gel electrophoresis. To avoid possible contamination, several measures, such as separate space to set up PCRs and disposable Wlter tips, were taken, as well a negative control (no DNA), and positive controls from diVerent strains of T. gondii were used. 2.7. RT-PCR for transcription analysis For RT-PCR assays, total RNA was puriWed from Toxoplasma tachyzoites using TRIzol (Invitrogen, USA) reagent. Sterile tubes, pipette tips, gloves, and diethyl pyrocarbonate treated water was used in extraction procedures. First-strand cDNA from RNA was synthesized by using Avian Myeloblastosis Virus (AMV) reverse transcriptase from Access RT-PCR system (Promega, Madison, WI). The reaction was performed in a 45 l mixture containing a standard enzyme buVer supplied by the manufacturer: 10 l of RNA sample, downstream (Tgnos2 GCACACGCT CAACTAATTAC) and upstream primers (Tgnos1 CTGC TTGCCGTTTGTTTCG) at 1.8 M concentration/0.2 mM each dNTPs and 0.1 U/l of the enzyme. The reaction mix-
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ture was incubated at 48 °C for 45 min and then at 94 °C for 2 min to inactive AMV enzyme. 5 l of the mixture was used for PCR analysis as a template cDNA. The PCR ampliWcation was performed in 100 l reaction mixtures containing 1 £ PCR buVer supplied by the manufacturer (PCR Master Mix, Promega, USA), 2.5 mM MgCl2, 200 M dNTPs, 2.5 U of recombinant Taq DNA polymerase, each of the primers at 1.0 M, and template cDNA. The Wrst step of ampliWcation was 30 s of denaturation at 94 °C. This step was followed by 15–40 cycles, with 1 cycle consisting of 30 s at 94 °C, 30 s at the 58 °C annealing temperature for each primer, and 30 s at 72 °C. The Wnal cycle was followed by an extension step of 10 min at 72 °C. RTPCR products were runned on 2% agarose electrophoresis gels and then visualized in an ultraviolet lamp. As a control in cDNA template for a housekeeping gene in tachyzoite stages we use tubulin primers (sense: AGTCCAGCGTCTGTGACATCC; antisense: GCACCCATCTCGCCCTCTTCC) and we performed 40 PCR cycles with 1 cycle consisting of 30 s at 94 °C for denaturation, 1 min at 62 °C for annealing, and a Wnal extension step of 1 min at 72 °C. 3. Results 3.1. NO inhibitor signiWcantly increase T. gondii growth in THP1 cells and the nitrites production (NO derivatives) of Toxoplasma During experiments on eVector protective mechanisms of IFN plus bacterial LPS the overall growth of intracellular Toxoplasma was increased in the presence of 1 M of the NO inhibitor NG monomethyl-L-arginine (NGMMA). This was signiWcantly reduced by IFN + LPS (Fig. 1). To understand the enhanced growth of Toxoplasma in the presence of NGMMA, the levels of nitrite (a stable NO derivative) were measured in supernatants of THP1 cell in the presence or not of Toxoplasma and treated or untreated with IFN plus LPS culture. IFN with LPS on THP1 culture cells, without the parasite, did not increase NO derivatives levels (Fig. 2). In contrast, Toxoplasma infection of THP1 cells (Fig. 2) and the supernatants of Toxoplasma maintained alone in saline solution (Fig. 3) produced 2–4 M of nitrites. The levels in supernatants were similar at concentration of 1 £ 106 or at 18 £ 106 tachyzoites concentration but NGMMA addition increased the nitrite concentration of tachyzoites alone in a linear fashion (Fig. 3). In addition, fractions of total lysate antigen (TLA) prepared after sonication of T. gondii tachyzoites or of secretory/excretory antigen (E/ S) with or without NGMMA, did not shown any signiWcant levels of nitrites (absorbance in TLA was 0.0 without NGMMA and 0.007 with NGMMA and in E/S was 0.0 without NGMMA and 0.0011 with NGMMA, these absorbancy values were below the level of detection of the Griess assay).
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Fig. 1. Overall growth of Toxoplasma in THP1 cells after 48 h of culture, calculated as the number of intracellular parasites by 100 cells in THP1 cultures in the absence (control) and in the presence of IFN 1000 U/ml plus LPS 10 g/ml (IFN + LPS) or in the absence or presence of 1 M NGMMA (+NGMMA). SigniWcant diVerences were found between (*) Control vs. IFN + LPS (t test p D 0.015) and between (**) NGMMA vs IFN + LPS + NGMMA (t test p D 0.000). The number of tachyzoites was determined by counting cells stained with a modiWed Giemsa stain. Bars represent means § SD of triplicates obtained in one experiment; similar results were obtained in three separate experiments.
Fig. 2. Nitrite levels in THP1 infected and treated or not with IFN plus LPS measured in THP1 cell culture supernatant by Griess reagent after 24 h of culture. Bars represent means § SE of triplicates (n D 9) of three separate experiments. Paired t test demonstrated statistically signiWcant diVerences in NO2 levels between Cell vs. Cell + T. gondii (Mann–Whitney nonparametric test p D 0.015). NaNO2 standard curve was from 0.05 to 500 M.
Fig. 3. Nitrite levels from Toxoplasma tachyzoites maintained alone in the presence or not of NGMMA and measured on saline solution supernatants after 24 h of culture. Nitrites levels were obtained by using Griess reagent and levels calculated by using a NaNO2 (Sigma) standard curve 0.05–500 M.
LLAVCFVSDFYSLSLLHFASVPFHESDGCVGRSH WLPGKHANYVKPAGARKRPEVGCRSSCLLRSVCC DILSPVRTRGNLSVCKYALVLSIACRGRSLLAGIQA LFEMPDLVTALIATMLFSGVLVIWNAWMCAAVLT IGRPVPKPLTSDAGEILGVETDFLAPAWRPVALGV VILSHLLQTETS. The translated aminoacid sequences have a predicted molecular weight of 20 kDa and include: a peptide signal, a transmembrane region, a glycosylation site, a N-myristoylation site and a NOS signature motif (Fig. 4). Multiple alignments of DNA sequences were performed with eNOS, iNOS, and nNOs sequences. Only the critical aminoacids are conserved in the NOS (C, G, and R residues) and in myristyl (V and C residues) motifs (Fig. 5). In NOS signature motif four residues are conserved along the phylogeny: C, G, R, and W. A phylogenetic tree placed Lymnea NOS motif sequence as the nearest sequence to the Toxoplasma NOS motif. Lymnea, Toxoplasma, and bacterial NOS are part of the same divergent
3.2. IdentiWcation of a NOS motif sequence in the Toxoplasma genome One deduced aminoacid genomic sequence of the contig TGG_994574 of ToxoDB, contained the motif NOS speciWc sequence motif [GR]C[IV]GR[ILS]W. We found, by using the Artemis software, a complete ORF sequence (561 bp) that begins at 152,107 and ends at 152,565 bp of the contig:
Fig. 4. Diagram of the protein domains in the Toxoplasma NOS sequence as obtained after submitting sequence to Prosite. Abbreviations, SP, Signal peptide; NOS, NOS motif; N-Myr, N-Myrystoil; Gly, Glycosylation; Tm, transmembrane domain. The sequence was obtained between 152,107 and 152,565 bp of the contig TGG_994574 of ToxoDB (toxodb.org).
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Fig. 5. Clustal multiple alignment of NOS sequences. (A) Multiple alignment with NOS of diVerent animal species; Boxes: * NOS motif, ** Myrystoil motif. (B) Residues pattern indicating the phylogenetic NOS motif (CXGRXXW). The sequences used for alignment were: gi冷266648冷 sp冷P29474冷 NOS3_HUMAN nitric oxide synthase, (endothelial NOS or eNOS); gi冷1352513冷 sp冷P35228冷 NS2A_HUMAN Nitric oxide synthase (inducible NOS or iNOS); gi冷1709333冷 sp冷P29475冷 NOS1_HUMAN nitric oxide synthase, brain (neuronal NOS or nNOS).
ancestor molecule that divide early into a group that incorporated a reductase domain (which includes the mammalian and metazoan NOS) and those that did not (Fig. 6). By using speciWc primers for a segment of the ORF, the PCR on genomic DNA obtained a 237 bp product. The molecular weight of the product was as predicted by in silico analysis (Fig. 7A). This sequence was ampliWed in many diVerent strains of Toxoplasma indicating that is conserved in the type I, type II, and type III lineages of Toxoplasma (Fig. 7B). 3.3. Putative NOS motif transcription RT-PCR analysis was performed to demonstrate that NOS motif was a transcription sequence. No DNA contamination was observed in Toxoplasma tachyzoite puriWed RNA. We used 33 g of Toxoplasma RNA to obtain cDNA. After 35 cycles, we found a plateau of PCR ampliWcation assays with the cDNA and we obtained a species speciWc 237 bp product (Fig. 8). The size of the product was as expected by the in silico analysis.
4. Discussion For many years the defensive role of NOS in human macrophages was a controversial topic, but it is now generally accepted that this is not an important mechanism in human toxoplasmosis (Gomez and Marin, 2000). Thus, levels of nitrites during activation of inducible NOS in mouse can reach 120 M or more but only 3–6 M in supernatant of human monocytes. Our experiments demonstrate that Toxoplasma, like other protozoan such as Tetrahymena (Christensen, 1996), Trypanosoma (Paveto et al., 1995), and Plasmodium (Ghigo et al., 1995), can have its own nitrite production that would reXect constitutive NOS activity (producing 2–6 M of nitrites). Genestra et al. (2003) have also shown that Leishmania produce levels of nitrite similar to those we found in Toxoplasma; however in their case, there was inhibition by several L-arginine analogs. We did not expected replication of Toxoplasma tachyzoites in medium without host cells. The experiment was performed over 24 h, which was long enough to keep Toxoplasma alive (Gómez Marin et al., 1996), and obtain nitrite production
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Fig. 6. Phylogenetic tree obtained using Mega software. Statistical method was the neighborhood joining tree and using 1000 bootstrapping replications.
by the parasite without nitrite production by host cells. We also measured levels of nitrites in parasite fractions (total antigen and excretory/secretory antigen) and since nitrites production was not found, indicating that NOS enzyme was not functional after this procedure of fractioning. We think that NOS activity in T. gondii was presumably induced (but not inhibited) by NGMMA, in live tachyzoites. Previous work has showed that NOS activity in THP1 cells is not a protective mechanism induced by IFN against Toxoplasma, and enhanced growth of T. gondii in the presence of NGMMA cannot be explained by an eVect on host NOS (Gomez and Marin, 2000). Nitrite production could be the result of many nitrogen consuming pathways (e.g., by the arginine deaminase enzyme activity). However, we consider, in the case of Toxoplasma, that nitrites production could also be the result of the NOS pathway. The existence of a putative NOS motif signature DNA sequence that is transcribed is a very strong argument to believe that nitrites in Toxoplasma would be derived from a NOS enzyme. The NOS motif [GR]C[IV]GR[ILS]W sequence that was used for the in silico search is the site where L-arginine bind to the eNOS enzyme. The sequence found in Toxoplasma was 561 bp long and has a low predicted molecular weight. The other regions predicted by the translated sequence, such as the N-Myrystoil site are important for the posttransductional membrane translation of the protein. The
membrane localization of the Toxoplasma NOS coded protein is also supported by the existence of the transmembrane domain. The presence of these motifs in the NOS sequence of Toxoplasma suggests that it is more related functionally to endothelial NOS (Raman et al., 1998). The NOS sequence in Toxoplasma is a primitive one as could be inferred by the phylogenetic analysis with the NOS sequences that are known currently. The NOS proteins included in the phylogenetic analysis showed silent mutations in the NOS motif such as a change of isoleucine to valine or to leucine (hidrophobes residues). Other residues changes such as the hydrophylic glutamine replaced by tirosine residues in mammals would be not as important as a change of glutamine to phenylalanine in bacterial NOS. The NOS conserved residues (C, G, R, and W) are evolutionary traces and deWnitive markers (phylogenetic motif) for the NOS protein family (Korneev and O’Shea, 2002). The phylogenetic tree suggests that nNOS and eNOS are the product of more ancillary gene duplication than those occurred for iNOS. Bacterial NOS is an independent branch that originated from this early duplication. One remarkable characteristic in mammals NOS is the presence of reductase domains that are absent in the Toxoplasma NOS and in bacterial NOS (Crane et al., 1998). One surprising Wnding was that one of best known NOS inhibitor increased the nitrites production by Toxoplasma.
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with the glutamate residue that binds to the L-arginine substrate (Crane et al., 1997). Kinetic studies with the puriWed NOS of Toxoplasma protein would be required to determine an allosteric eVect of NGMMA. Previously, NOS enzyme has been isolated from other protozoa; Basu et al. (1997) puriWed a NOS protein from Leishmania donovani. Present results encourage us to continue exploring the Toxoplasma NOS sequence as a probable therapeutic target; Wrst, the RT-PCR assays indicate that the putative NOS sequence is a transcriptionally active one. Second, NGMMA signiWcantly increases the growth of Toxoplasma in cell culture indicating that NOS it would have an important role stimulating parasite metabolism. As NGMMA has the opposite eVect on mammalian NOS, this suggests that Toxoplasma NOS has a unique structure that could permit the design of speciWc inhibitors without interference with mammalian NOS. In conclusion, we have identiWed the Wrst putative NOS coding DNA sequence in a protozoan pathogen; further studies are warranted to purify and to analyze its biochemical and functional characteristics. Fig. 7. Putative NOS PCR assay, genomic DNA was obtained from the tachyzoite stage of Toxoplasma. (A) MW: Molecular weight marker Puc18 digested with BamH1. 1:Escherichia coli DH5 strain. 2: Taenia solium. 3: Homo sapiens leukocytes. 4: T. gondii Rh strain. 5: Negative control (PCR mix without DNA). DNA concentrations of samples were checked by electrophoresis before PCR. (B) 1: Negative control (PCR mix without DNA). 2: DNA from Martin Toxoplasma strain (type I lineage), 3: SQM Toxoplasma strain (type II lineage), and 4: CEP Toxoplasma strain (type III lineage).
Acknowledgments Andres Gutierrez was beneWciary of a grant from the Young Researcher Program from Colciencias (Colombia). We thank specially to Aylan Arenas and Jorge Jimenez who collaborate in some Wnal experiments. Preliminary genomic and/or cDNA sequence data was accessed via http://ToxoDB.org and/or http://www.tigr.org/tdb/t_gondii/. Genomic data were provided by The Institute for Genomic Research (supported by the NIH Grant #AI05093), and by the Sanger Center (Wellcome Trust). EST sequences were generated by Washington University (NIH Grant #1R01AI045806-01A1). We thank Dr. Herney Bolivar who checked our English spelling. References
Fig. 8. RT-PCR assay for putative NOS sequence. MW: Molecular weight marker Puc18 digested with BamH1. 1: Toxoplasma tubulin ampliWed by using the speciWc primers as described in Section 2. 2: SpeciWc ampliWcation of putative NOS sequence from cDNA of T. gondii RH strain tachyzoite. 3 and 4: RT negative control to rule out contaminating DNA was performed by using a RT reaction product without reverse transcriptase during cDNA synthesis.
NGMMA is L-arginine structural analog and inhibits NOS in mammals by competing with binding to the NOS motif of L-arginine. Crystallographic studies have found that NGMMA and other NOS speciWc inhibitors like aminoguanidine, S-ethylthiourea and thiocitruline directly interacts
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