S-Adenosylmethionine Decarboxylase from Leishmania donovani

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5 -(((Z)4-amino-2-butenyl)methylamino)-5 -deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a devastating and often fatal disease in humans. The parasite exhibits a digenetic life cycle with the extracellular promastigote residing in the phlebotomine sandfly vector and the intracellular amastigote propagating within the phagolysosome of mammalian macrophages. Because no effective vaccine for leishmaniasis is available, chemotherapy offers the only means of disease treatment. However, the current arsenal of drugs for treating leishmaniasis is far from ideal because these compounds are moderately to highly toxic, the result of their lack of target specificity. Recently, the emergence of drug-resistant strains has exacerbated the need for more selective and efficacious drugs to treat or prevent leishmaniasis or, for that matter, many other parasitic diseases. One pathway that has been exploited successfully in antiparasitic drug regimens is that for the synthesis of polyamines, organic cations that play indispensable roles in key cellular processes such as growth, differentiation, and macromolecular biosynthesis (1, 2). D,L-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, can eradicate Trypanosoma brucei infections in both mice (3) and patients with late stage African sleeping sickness (4, 5). The selective window for drug efficacy is not achieved, however, by dissimilar DFMO binding affinities for the T. brucei and human ODC enzymes but rather to differences in enzyme stability (6–8). DFMO is also active against many other protozoan parasites, including the promastigote form of L. donovani (9–11). Another inhibitor of the polyamine pathway, 5 -(((Z)-4-amino-2-butenyl)methylamino)-5 -deoxyadenosine (MDL 73811), is also effective in eradicating T. brucei infections in mice (12, 13). MDL 73811 is an irreversible inhibitor of ADOMETDC, the enzyme that catalyzes the irreversible decarboxylation of Sadenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet) substrate for spermidine synthase (SPDSYN). In addition to MDL 73811, ADOMETDC is also inhibited in vitro by a battery of other trypanocidal drugs, including pentamidine, berenil, and methylglyoxyl bis* This work was supported in part by Grants AI10096 (to S. C. R.) and AI41622 (to B. U.) from the NIAID, National Institutes of Health, by grants from the Swedish Natural Science Research Council, the J. C. Kempe Memorial Foundation, and the Royal Physiographic Society in Lund (to O. H.), and by a grant from the Burroughs Wellcome Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) U20091. § Present address: Community Blood Centers of Florida, 1700 N. State Road 7, Lauderhill, FL 33313. ¶ Present address: Institute for Hygiene, Dept. of Virology, University of Heidelberg, 69120 Heidelberg, Germany. Present address: Corixa Corporation, 1124 Columbia St., Seattle, WA 98104. ** Present address: McGill University, MacDonald Campus, 21,111 Lakeshore Rd., Ste. Anne Bellevue, Quebec, Canada, H9X 3V9. §§ Burroughs Wellcome Fund scholar in molecular parasitology. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, L224, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-8437; Fax: 503-494-8393; E-mail: ullmanb@ohsu.edu. 1 The abbreviations used are: DFMO, D,L-difluoromethylornithine; ODC, ornithine decarboxylase; MDL 73811, 5 -(((Z)-4-amino-2-butenyl) methylamino)-5 -deoxyadenosine; ADOMETDC, S-adenosylmethionine decarboxylase; AdoMet, S-adenosylmethionine; dAdoMet, decarboxylated S-adenosylmethionine; SPDSYN, spermidine synthase; MGBG, methylglyoxyl bis(guanylhydrazone); MTA, 5-methylthioadenosine; DME-L, Dulbecco’s modified Eagle-based medium designed for Leishmania cultivation; DME-L-CS, DME-L, in which the bovine serum albumin component was replaced with chicken serum; ORF, open reading frame; 5 F, 5 -flanking region; 3 F, 3 -flanking region. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 8, Issue of February 22, pp. 5902–5909, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a devastating and often fatal disease in humans.The parasite exhibits a digenetic life cycle with the extracellular promastigote residing in the phlebotomine sandfly vector and the intracellular amastigote propagating within the phagolysosome of mammalian macrophages.Because no effective vaccine for leishmaniasis is available, chemotherapy offers the only means of disease treatment.However, the current arsenal of drugs for treating leishmaniasis is far from ideal because these compounds are moderately to highly toxic, the result of their lack of target specificity.Recently, the emergence of drug-resistant strains has exacerbated the need for more selective and efficacious drugs to treat or prevent leishmaniasis or, for that matter, many other parasitic diseases.
One pathway that has been exploited successfully in antiparasitic drug regimens is that for the synthesis of polyamines, organic cations that play indispensable roles in key cellular processes such as growth, differentiation, and macromolecular biosynthesis (1, 2).D,L-␣-Difluoromethylornithine (DFMO), 1 an irreversible inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, can eradicate Trypanosoma brucei infections in both mice (3) and patients with late stage African sleeping sickness (4,5).The selective window for drug efficacy is not achieved, however, by dissimilar DFMO binding affinities for the T. brucei and human ODC enzymes but rather to differences in enzyme stability (6 -8).DFMO is also active against many other protozoan parasites, including the promastigote form of L. donovani (9 -11).
(guanylhydrazone) (MGBG).Pentamidine is also the second most prescribed drug for treatment of visceral leishmaniasis.However, even though the T. brucei ADOMETDC enzyme is inhibited by each of these compounds, the intracellular target for these drugs has not been established.Indeed, other cellular targets for pentamidine and berenil have been proposed (14 -17), and the potential lack of target specificity may also contribute to the host toxicity observed with these compounds.
To determine whether ADOMETDC inhibition is a valid therapeutic paradigm and to elucidate the cellular targets of inhibitors of ADOMETDC in a model and genetically tractable parasite, the ADOMETDC gene and its flanking sequences have been isolated from L. donovani.These DNAs were used to generate ⌬adometdc knockouts employing double targeted gene replacement strategies (18 -20) and ADOMETDC overexpressors.The ⌬adometdc L. donovani were shown to be auxotrophic for spermidine, and incubation of these parasites in the absence of exogenous polyamines resulted in a marked depletion in the cellular pools of both spermidine and trypanothione, a spermidine-containing thiol conjugate.Furthermore, the ADOMETDC overproducers were resistant to MDL 73811 but not to the other ADOMETDC inhibitors, establishing that the enzyme is the primary intracellular target for only one of the four antiparasitic drugs that target the enzyme in vitro.

EXPERIMENTAL PROCEDURES
Materials, Chemicals, and Reagents-The pX63NEO, pX63HYG, pX63PAC, and pXNEO plasmids used in the transfection experiments were provided by Dr. Stephen M. Beverley (Washington University, St. Louis, MO).Spermidine, putrescine, MTA, berenil, pentamidine, and MGBG were obtained from Sigma.Hygromycin was procured from Roche Molecular Biochemicals, G418 was purchased from BioWhittaker, and MDL 73811 was a gift from the now defunct Marion Merrell Dow Research Institute.
Cell Culture-The wild type L. donovani clone, DI700, originally derived from the 1S Sudanese strain, was used for DNA isolation, library construction, and as a recipient strain in the initial transfections.L. donovani promastigotes were propagated in DME-L, a completely defined Dulbecco's modified Eagle-based medium especially designed for Leishmania cultivation (21).Transfected parasites were maintained in a modified medium, DME-L-CS, in which the bovine serum albumin component was replaced with 10% chicken serum to avoid polyamine oxidase-mediated toxicity (11).The ADOMETDC/ adometdc heterozygote and the ⌬adometdc null mutants were routinely maintained in DME-L-CS supplemented with either 50 g/ml hygromycin or 50 g/ml hygromycin, 20 g/ml G418, and 100 M spermidine, respectively, unless otherwise specified.The ADOMETDC overexpressor, DI700[pADOMETDC], and DI700 parasites transfected with just pXNEO, DI700[pXNEO], were grown in DME-L-CS and 20 g/ml G418.The ⌬adometdc null mutants transfected with pX63PAC[ADOMETDC], designated ⌬adometdc[pADOMETDC], were propagated in DME-L-CS to which 50 g/ml hygromycin, 20 g/ml G418, and 100 M puromycin were added.Growth rate experiments were conducted and parasites enumerated on a hemacytometer (Hausser Scientific Co.) or a Coulter Counter model ZF as described (11,19).
Isolation of the ADOMETDC-A fragment of ADOMETDC was amplified from genomic DNA using PCR.The sense primer, 5Ј-CTCG-GAATTCC-TT[CT]-GAG-GG[CGT]-[AC]C[CG]-GAG-AA-3Ј, was constructed with an 11-nucleotide leader encompassing an EcoRI site (underlined) followed by a degenerate sequence corresponding to amino acids 7-12, FEGTEK, of the human ADOMETDC, whereas the antisense primer, 5Ј-CTCGGGATCCC-CTC-[ACG]GG-[CG]GT-[AG]AT-GT-G-3Ј, was generated from residues 243-248, HITPEP of the human ADOMETDC and was preceded by an 11-nucleotide leader encompassing a BamHI restriction site (underlined).Amplification of the ADOMETDC fragment was performed on a Coy Instruments thermocycler using the PCR conditions described by Hanson et al. (22).The resulting 720-bp fragment was sequenced according to the dideoxy termination method and used to screen an L. donovani EMBL3 genomic library (23) under stringent hybridization and washing conditions (22,23).A 5.1-kb SalI fragment that hybridized to the probe was subcloned from a plaque-purified phage into pBluescript KSϩ, and the entire nucleotide sequence of the ADOMETDC open reading frame (ORF) was obtained in both orientations.In addition, portions of the ADOMETDC 5Ј-and 3Ј-flanking regions were also sequenced.Multiple sequence alignments with ADOMETDC proteins from phylogenetically diverse organisms were performed using the Feng-Doolittle algorithm (24).
Antibody Generation and Characterization-To raise antibodies against ADOMETDC, the ADOMETDC gene was inserted into the pBAce prokaryotic expression vector and then induced in low phosphate induction medium as described (25).The protein was recovered in virtually pure form as inclusion bodies and employed as immunogen to generate polyclonal antibodies in rabbits (Cocalico Biologicals Inc.) using standard injection protocols.Immunoblot analysis was carried out according to standard protocols (26,27).
Molecular Constructs for the Replacement of the ADOMETDC Alleles-To construct drug resistance cassettes to replace both ADOMETDC alleles, ϳ300 bp of 5Ј-and ϳ1 kb of 3Ј-flanking regions were subcloned into the appropriate sites of the pX63HYG and pX63NEO plasmids.The 5Ј-flanking region, 5ЈF, was amplified by PCR using the M13-20 oligonucleotide (containing pBluescript sequence) as a sense primer and an antisense primer, 5Ј-GGGAAAAGCGTCGACAA-GAGGCGATGAGC-3Ј, preceded by an SalI site (underlined) and corresponding to a region ϳ120 nucleotides upstream from the ADOMETDC ORF.The ϳ1,200-bp PCR product was digested with SalI and HindIII, a restriction site located ϳ300 bp upstream from the SalI site, and 5ЈF was then subcloned into the HindIII/SalI sites of pX63HYG and pX63NEO.The resulting plasmids were termed pX63HYG-5ЈF and pX63NEO-5ЈF, respectively.To amplify the 3Ј-flank, a sense primer, 5Ј-GTCCCCGGGCCGCCCCCCTTATCCCC-3Ј, and an antisense primer, 5Ј-CTCAGATCTCCCTTTAGTGAGGG-3Ј, containing SmaI and BglII restriction sites (underlined), respectively, were designed.The ϳ1-kb PCR product, 3ЈF, was then subcloned into the corresponding sites of pX63HYG-5ЈF and pX63NEO-5ЈF to yield the knockout constructs, pX63NEO⌬adometdc and pX63HYG⌬adometdc, respectively.The correct orientation of 5ЈF and 3ЈF within the two drug resistance cassettes was confirmed by restriction enzyme digestion and limited nucleotide sequencing.
ADOMETDC Overexpression Vectors-The ADOMETDC ORF was amplified by PCR and cloned into the pXNEO and pX63PAC vectors to create pXNEO[ADOMETDC] and pX63PAC[ADOMETDC], respectively.Oligonucleotides used for the construction of pXNEO[AD-OMETDC] were 5Ј-TCTGGATCCAGCGGCTCTGAGGCG-3Ј (BamHI site underlined) for the sense primer and 5Ј-TCCTCTAGAGTGATGAC-CTTTGCG-3Ј (XbaI site underlined) for the antisense primer.The PCR product was then subcloned into the BamHI and XbaI sites of the leishmanial expression vector pXNEO to create pXNEO[ADOMETDC].To amplify the ADOMETDC ORF for insertion into pX63PAC, both the sense primer, 5Ј-GCAGGATCCACCATGAATGTCGC-3Ј, and the antisense primer, 5Ј-CGCGGATCCAAGAACTAGTCGGGCC-3Ј, contained BamHI restriction sites.The PCR product was then subcloned into the BamHI site of pX63PAC to create pX63PAC[ADOMETDC], and the correct orientation was ascertained by further restriction enzyme digestion.
Transfections-The ⌬adometdc null strains were generated from wild type L. donovani by double targeted gene replacement.The pX63HYG⌬adometdc and pX63NEO⌬adometdc plasmids were digested with HindIII and BglII, and the fragments containing the ADOMETDC flanking regions and drug resistance markers, designated HYG⌬adometdc and NEO⌬adometdc, respectively, were gel purified and transfected into wild type parasites and ADOMETDC/adometdc heterozygotes, respectively, using previously reported electroporation conditions (19,28).Clonal ADOMETDC/adometdc heterozygotes were selected on plates in semisolid DME-L-CS medium containing 50 g/ml hygromycin, and the genotypes were confirmed by Southern blotting.Clonal ⌬adometdc knockout lines were then obtained from a heterozygote clone after a second round of transfection and plating in DME-L-CS medium supplemented with 100 M spermidine, 100 M MTA, 50 g/ml hygromycin, and 20 g/ml G418.The genotypes of the null mutants were again verified by Southern blotting.

RESULTS
Isolation of the ADOMETDC Gene-The gene encoding ADOMETDC was isolated on a 5.1-kb SalI fragment using a 720-bp ADOMETDC PCR fragment as a hybridization probe.Nucleotide sequence analysis identified a 1,149-bp ORF encoding a protein of 382 amino acids with a molecular mass of 44.2 kDa for the proenzyme and 33.1 and 11.1 kDa for the ␣ and ␤ subunits, respectively.An in-frame termination codon was located 27 nucleotides upstream from the assigned initiation codon.In pairwise alignments, ADOMETDC exhibited amino acid identities of 70, 62, 30, 30, and 28% with the Trypanosoma cruzi (29, 30), T. brucei (accession number AAA61969), human (31), Onchocerca volvulus (32), and Saccharomyces cerevisiae (33) ADOMETDC proteins, respectively.A multisequence alignment revealed several regions of high homology, most notably in the putative proenzyme cleavage site between amino acids Glu-90 and Ser-91 of the L. donovani enzyme (34, 35) (Fig. 1).Southern blot analysis of L. donovani genomic DNA demonstrated that ADOMETDC is a single copy gene, and Northern blots revealed the existence of a single ADOMETDC 1.8-kb transcript in L. donovani promastigotes (data not shown).
Stability of the ADOMETDC Protein-Because the mammalian ADOMETDC is known to turn over very rapidly (the half-life is Ͻ1 h) (36)(37)(38), the stability of ADOMETDC in L. donovani promastigotes was examined.Unlike the mammalian enzyme, however, ADOMETDC is stable up to 24 h after treatment with cycloheximide at a concentration that obliterates protein synthesis in L. donovani (22).Indeed, the amount of ADOMETDC protein after 24 h of cycloheximide treatment is equivalent to that of exponentially growing parasites (Fig. 2).
Construction and Molecular Characterization of ⌬adometdc Null Mutants-To disrupt the ADOMETDC locus in L. donovani, each gene copy was sequentially replaced with a drug resistance cassette.The first ADOMETDC copy was replaced with pX63HYG⌬adometdc, and clonal ADOMETDC/adometdc heterozygotes were selected in hygromycin.A second round of transfection with pX63NEO⌬adometdc replaced the second ADOMETDC allele, and clonal strains of ⌬adometdc knockout parasites were selected in hygromycin, G418, MTA, and spermidine.The presence of hygromycin during the second round of gene targeting ensured that the pX63NEO⌬adometdc had replaced the wild type allele in the ADOMETDC/adometdc heterozygotes, whereas spermidine and MTA were added to the selective medium to bypass any potential auxotrophy.Approximately 16 colonies were picked from plates after the second round of gene targeting.After initial screenings, two of these clones, ⌬adometdc13 and ⌬adometdc14, were selected for further analysis.
Southern blot analysis of SalI-digested genomic DNA prepared from wild type, ADOMETDC/adometdc, ⌬adometdc13, and ⌬adometdc14 parasites hybridized to the ADOMETDC ORF clearly demonstrated the loss of both ADOMETDC copies in the knockout lines (Fig. 3, panel A).Southern blot analysis of SacI-cut DNA and probing with the ADOMETDC 5Ј-and 3Јflanking regions, 5ЈF and 3ЈF, respectively, revealed the precise nature of the rearrangements observed after the homologous recombination events that gave rise to the heterozygous and homozygous genotypes.The creation of the novel and loss of the wild type alleles are unveiled by probing with either 5ЈF (Fig. 3, panel B) or 3ЈF (Fig. 3, panel C).Replacement of the wild type ADOMETDC copies by HYG⌬adometdc and NEO⌬adometdc created an additional 3.8-kb SacI fragment that hybridized to 5ЈF (Fig. 3, panel B) and a 5.5-kb SacI fragment recognized by 3ЈF (Fig. 3, panel C).The loss of the remaining wild type allele from the heterozygote is apparent after the second round of transfection with NEO⌬adometdc in both knockout lines (Fig. 3, panels B and C).Partial restriction maps of the wild type and replaced loci are depicted in Fig. 3, panel D. It is worth noting that 3ЈF also recognizes other sequences within the L. donovani genome and gives rise to the unexpected background signals in Fig. 3, panel C.These signals were unaffected by any of the recombination events at the ADOMETDC locus.
The loss of the wild type ADOMETDC copies was corroborated via Western blot analysis of ADOMETDC overproducer, wild type, ⌬adometdc13 and ⌬adometdc14 lysates (Fig. 4).Whereas the polyclonal antibodies recognize a single polypeptide of ϳ35 kDa, no band is observed in either of the knockout strains.This band presumably corresponds to the ␣ subunit of ADOMETDC because it is consistent with the predicted molecular mass from the translated nucleotide sequence.The antibodies do not appear to recognize the ␤ subunit of the enzyme with sufficient avidity for visualization by immunoblotting.Equal loading of protein lysates from wild type and knockout strains onto the gel was confirmed by normalization of the immunoblots to antisera raised against the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase.
Nutritional Requirements of the ⌬adometdc Strains-To evaluate the nutritional requirements of the ⌬adometdc FIG. 2. Stability of the L. donovani ADOMETDC protein.Wild type parasites were treated with 500 mg/ml cycloheximide, and samples for Western blot analysis were taken before the addition of cycloheximide (0 h) and 1, 2, 3, 4, 6, and 24 h after the addition of cycloheximide.The Western blot was then probed with the polyclonal antibodies raised against L. donovani ADOMETDC.The bands were quantitated by scanning on a densitometer, and the signal intensities relative to the zero point control are indicated below the blot.This experiment was repeated one additional time with essentially equivalent results.strains, the knockout parasites were incubated in growth medium with and without polyamines or MTA.As shown in Fig. 5A, wild type and ADOMETDC/adometdc grown in defined DME-L-CS without polyamine supplementation grew equally well, whereas the ⌬adometdc13 and ⌬adometdc14 cells did not proliferate at all.The null mutants incubated under these conditions arrested after three to four cell divisions, assumed a rounded morphology, and died after 4 weeks.The addition of 100 M spermidine was able to circumvent the phenotypic consequences of the genetic lesion in ⌬adometdc parasites, whereas an equivalent concentration of putrescine or MTA, a product of the Leishmania dADOMETC reaction, did not rescue the parasites (Fig. 5B).Finally, the growth defect of ⌬adometdc parasites was also not restored by the addition of 100 M spermine, a polyamine that is not found in L. donovani (19), to the culture medium (data not shown).The failure of these compounds to rescue the knockouts could not be imputed to their inherent toxicity because none of these compounds adversely affected the viability or growth of wild type L. donovani (data not shown).The ability of spermidine to circumvent the polyamine auxotrophy of ⌬adometdc parasites demonstrates that the sole cellular function of dAdoMet is to serve as an aminopropyl donor in spermidine synthesis and that MTA production by this pathway is nonessential for L. donovani promastigotes.
Genetic Rescue of ⌬adometdc Mutants-To establish that the auxotrophic phenotype observed in the knockout parasites could be ascribed to the genetic lesions at the ADOMETDC locus, an episomal copy of the ADOMETDC gene was introduced into the ⌬adometdc14 line by transfection and amplified in the selective drug.As shown in Fig. 6A, two different clonal populations of ⌬adometdc[pADOMETDC] parasites contained levels of ADOMETDC protein significantly greater than that of wild type parasites (Fig. 6A).Furthermore, complementation of the knockout strain restored the ability of the parasites to proliferate in polyamine-deficient medium (Fig. 6B).In fact, the growth rate was slightly greater than that of wild type parasites.
Polyamine, Thiol, and dAdoMet Pools-To evaluate the consequences of polyamine starvation on the intracellular polyamine and reduced thiol pools, ⌬adometdc14 parasites were

FIG. 5. Growth of wild type and mutant parasites in DME-L-CS medium in the absence and presence of polyamine supplements.
Panel A, wild type (f), ADOMETDC/adometdc (q), ⌬adometdc13 (OE), and ⌬adometdc14 () were grown in DME-L-CS medium with no polyamine additions.Parasites were enumerated every 24 h by counting in a hemacytometer.Panel B, the ⌬adometdc14 parasites were removed from DME-L-CS medium that had been supplemented with 100 M spermidine, washed twice in phosphate-buffered saline, and then incubated in medium without polyamine addition for 4 days and subsequently incubated in medium with no supplementation (‚) or supplemented with either 100 M spermidine (ࡗ), MTA (ƒ), or putrescine (OE).incubated in polyamine-free medium for 8 days.During this incubation, when parasites were not dividing, the intracellular levels of spermidine decreased significantly with a concomitant increase in the intracellular putrescine pool (Fig. 7A).The putrescine:spermidine ratio increased from an initial 1.4 prior to the experiment to a final ratio of 60 after 8 days of incubation in the polyamine-deficient DME-L-CS.The absolute concentrations of both polyamines were similar in wild type (10 nmol of putrescine/10 8 cells and 10 nmol of spermidine/10 8 cells) and ⌬adometdc14 parasites when the latter were maintained in spermidine (16 nmol of putrescine/10 8 cells and 6 nmol of spermidine/10 8 cells).Concurrent with the spermidine depletion was a diminution in the level of reduced trypanothione, a spermidine-containing thiol conjugate, and an increase in the pool of glutathione, the thiol precursor for trypanothione synthesis (Fig. 7B).Although trypanothione levels were 4-fold higher than glutathione levels in the ⌬adometdc14 parasites that had been maintained in spermidine-supplemented growth medium, the levels of trypanothione and glutathione were equivalent after 2 days of polyamine starvation, and the glutathione pool was 27-fold greater than that of trypanothione after 8 days of incubation in the polyamine-deficient medium.The thiol pools of wild type and ⌬adometdc14 parasites grown in spermidine-supplemented DME-L-CS were roughly equivalent (data not shown).Finally, as expected, dAdoMet was not detected in the ⌬adometdc14 cells.
ADOMETDC Inhibition Profiles-To evaluate whether ADOMETDC was the intracellular target of a variety of known inhibitors of ADOMETDC enzymes in vitro, the effects of these enzyme inhibitors were compared on wild type and DI700-[pADOMETDC] parasites that overexpressed ADOMETDC (Fig. 8 and Table I).As shown in Fig. 8, the ADOMETDC overproducer exhibited significant resistance to MDL 73811.Whereas the effective concentration of MDL 73811 which inhibited growth of wild type parasites by 50% (EC 50 value) was ϳ40 M, the parasites that overproduced ADOMETDC were virtually unaffected by the presence of 10 mM MDL 73811 in the culture medium.Addition of spermidine conferred refractoriness of wild type cells to MDL 73811 as well (Fig. 8).In contrast, the EC 50 values of wild type and DI700[pADOMETDC] parasites for berenil, pentamidine, and MGBG were equivalent (Table I).Spermidine addition did not influence the EC 50 values of wild type parasites for any of the last three compounds.

DISCUSSION
The construction of ⌬adometdc parasites by double targeted gene replacement established that ADOMETDC is an essential gene in L. donovani promastigotes.The ⌬adometdc parasites were shown to be polyamine auxotrophs, and this auxotrophy could only be rescued by spermidine supplementation of the culture medium or by complementation with an episomal copy of ADOMETDC.The ability of spermidine to circumvent the polyamine auxotrophy of ⌬adometdc parasites demonstrates that the sole cellular function of dAdoMet is to serve as an aminopropyl donor in spermidine synthesis.The growth phenotype and nutritional requirements of the ⌬adometdc cells parallel those of previously described ⌬spdsyn L. donovani (20).Conversely, a ⌬odc knockout could also be rescued by spermidine, but only at high concentrations where growth rates were suboptimal (19).Thus, the growth phenotypes of the knockout lines demonstrate that spermidine is both essential and sufficient for promastigote survival.In contrast, putrescine, which is synthesized in both ⌬adometdc and ⌬spdsyn, is unable to meet the entire polyamine requirement of the parasite.However, ⌬odc parasites grow better in medium supplemented with putrescine than with spermidine alone, suggesting that the diamine is essential for optimal growth and is, therefore, more than just a precursor for spermidine synthesis.The inability of spermine, a ligand for the polyamine transporter (39), to circumvent a genetic lesion in any of the three polyamine biosynthetic enzymes implies that L. donovani promastigotes lack a mechanism to convert spermine to spermidine and spermidine to putrescine.
The precise mechanism by which a lesion in ADOMETDC precipitates a failure to thrive in the absence of spermidine is not clear.Although spermidine is essential for cell proliferation in higher eukaryotes that do not make trypanothione, the spermidine-conjugate is postulated to be vital for the maintenance of the redox potential in Leishmania (40,41).Incubation of ⌬adometdc cells in medium lacking spermidine resulted in a dramatic decrease in the intracellular pools of both spermidine and trypanothione with a coincident augmentation of putrescine and glutathione levels.The effects of the ⌬adometdc lesion on polyamine and thiol pools mirror those obtained with ⌬spdsyn L. donovani, although dAdoMet pools were undetectable in ⌬adometdc but markedly elevated in ⌬spdsyn cells (20).
The buildup of both putrescine and glutathione pools under polyamine starvation conditions (Fig. 7) may be the result of cellular overproduction of these two metabolites to compensate for the genetically induced spermidine and trypanothione deficiencies.In support of this hypothesis is the observation that ⌬adometdc parasites supplemented with spermidine maintain putrescine and glutathione levels that are roughly equivalent to those of wild type parasites.Thus, spermidine and trypanothione levels somehow influence the synthesis of their precursors within the parasite by one or more unknown regulatory mechanisms.Obviously, however, the increases in putrescine and glutathione observed in the polyamine-starved ⌬adometdc parasites do not ultimately compensate for the loss of spermidine and/or trypanothione.
ADOMETDC has received considerable attention as a potential antiparasitic drug target.Pentamidine, berenil, and MGBG, structural analogs of spermidine, and MDL 73811, a dAdoMet analog, are toxic toward L. donovani (Table I) and T. brucei (12,42,43), and each inhibits the mammalian and T. brucei ADOMETDC activities in vitro (42,44,45).However, all four inhibitors have a multitude of potential targets, and it remains unknown whether their cytotoxic effects can be ascribed to ADOMETDC inhibition (14, 15, 42, 46 -48).To evaluate whether ADOMETDC was the primary intracellular target for these drugs in L. donovani, an ADOMETDC overproducer was generated.Comparisons of the drug profiles of wild type and the DI700[pADOMETDC] overproducers revealed equal sensitivities of both lines to pentamidine, berenil, and MGBG, whereas the DI700[pADOMETDC] parasites were dramatically resistant to the growth inhibitory and cytotoxic effects of MDL 73811 (Fig. 8 and Table I).The pharmacogenetic experiments are supported by the ability of spermidine to circumvent the toxicity of MDL 73811, but not of pentamidine, berenil, and MGBG, in wild type parasites.Thus, ADOMETDC is the primary cellular target for MDL 73811 in L. donovani promastigotes, whereas the other three drugs do not exert their toxicity via ADOMETDC inhibition.
A multisequence alignment of ADOMETDC proteins from phylogenetically diverse organisms revealed several regions of significant sequence homology.ADOMETDC maintains the conserved cleavage site between Glu-90 and Ser-91 which con-verts the apoenzyme into mature ␣ and ␤ subunits.Western blot analysis implies that the L. donovani ADOMETDC translation product is cleaved in the parasite and is present predominantly as the mature enzyme because the antibodies appear to recognize primarily the 33-kDa ␣ subunit (Fig. 4).However, the 44-kDa apoenzyme can be observed as a very faint band in both ⌬adometdc[pADOMETDC] (Fig. 6) and DI700[pADOMETDC] overexpressors (data not shown).In addition, virtually all of the residues that were identified by structural and genetic analyses to be critical for catalysis, ligand binding, and proenzyme processing of the human ADOMETDC are conserved in the L. donovani enzyme.These include (i) Cys-105, Ser-255, and His-268 of the L. donovani enzyme, which correspond to Cys-82, Ser-229, and His-243 of the human ADOMETDC and which are essential for catalysis; (ii) Phe-32, Leu-87, Phe-249, and Glu-271 of the L. donovani ADOMETDC, which match Phe-7, Leu-65, Phe-223, and Glu-247 of the human counterpart and which are necessary for proper ligand positioning and binding; and (iii) Glu-36, Asp-195, His-268, and Glu-280 of the parasite enzyme, which align with Glu-11, Asp-174, His-243, and Glu-256 of the human protein, which are crucial for ADOMETDC processing (49 -52).
The mammalian ADOMETDC enzyme is regulated by several different mechanisms.One primary control mechanism is the rapid turnover rate (36 -38), a feature not shared by the leishmanial counterpart (Fig. 2).The L. donovani ODC (22) and SPDSYN enzymes are also stable.In contrast, ODC of mammalian cells is among the most labile of cellular proteins (53,54), whereas the mammalian SPDSYN is quite stable (55).The stability of the parasite ADOMETDC and the lability of the host equivalent may account for the ability of MDL 73811 to eradicate mouse infections of the related parasite T. brucei.Although both the mammalian and parasite ADOMETDCs are inhibited by MDL 73811 (56) (Fig. 8 and Table I), the ability of the host cell enzyme to recover rapidly from the pharmacological insult may account for the therapeutic selectivity of the drug (12,43).A similar paradigm has been proposed and substantiated experimentally for the efficacy of DFMO on T. brucei infections (57).Although both mammalian and T. brucei ODC enzymes are equally sensitive to DFMO inhibition, the host cell, unlike the parasite, has the ability to regenerate ODC quickly after the drug has been eliminated.
These genetic studies have demonstrated that ADOMETDC is an essential protein in L. donovani promastigotes and that spermidine is an essential polyamine.The existence of ADOMETDC inhibitors that pharmacologically simulate a genetic deficiency in the enzyme, the disparate stabilities of the mammalian and parasite ADOMETDC proteins, and the previously determined biochemical discrepancies between the mammalian and leishmanial polyamine pathways imply that ADOMETDC may be a potential target for therapeutic manipulation of certain parasitic diseases.

FIG. 3 .
FIG. 3. Southern blot analysis of wild type and mutant parasites.Panels A, B, and C, 2 g of genomic DNA from wild type, ADOMETDC/adometdc, ⌬adometdc13, and ⌬adometdc14 parasites was digested with the indicated restriction enzymes and hybridized to ADOMETDC probes from the coding region (panel A), the 5ЈF (panel B), or the 3ЈF (panel C).Molecular weight markers are indicated to the left.Panel D, restriction maps of the wild type ADOMETDC and novel loci are depicted.A black oval shows the ADOMETDC coding region probe, hatched boxes indicate both flanking regions, and a dotted box indicates the HYG or NEO locus.

FIG. 7 .FIG. 8 .
FIG. 7. Polyamine and thiol pool measurements in ⌬adometdc parasites.The ⌬adometdc14 parasites were incubated in DME-L-CS without polyamine addition, and aliquots were analyzed every 2 days for putrescine (f) and spermidine (ࡗ) in panel A, and glutathione (q) and trypanothione (OE) in panel B.