From the Department of Biochemistry and Molecular
Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098 and ¶ Department of Biochemistry, University of Dundee, Dundee DD1
4HN, Scotland, United Kingdom
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ABSTRACT |
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A knockout strain of Leishmania
donovani lacking both ornithine decarboxylase (ODC) alleles has
been created by targeted gene replacement. Growth of Polyamines are cationic compounds that play essential roles in
cell proliferation, differentiation, and macromolecular synthesis (1-3). Ornithine decarboxylase
(ODC)1 catalyzes the
conversion of ornithine to putrescine (1,4-diaminobutane) and is the
initial and rate-limiting enzyme in polyamine biosynthesis in most
organisms (4). The ODC enzyme of protozoan parasites is a novel
therapeutic target, because
D,L- The ability of trypanosomatids to undergo a very high frequency of
homologous recombination allows the disruption of chromosomal loci with
transfected drug resistance cassettes (11, 12) and permits a direct
test of gene function. This enables the creation of conditionally
lethal parasite strains whose survival and ability to propagate are
dependent upon the provision of compounds that can ameliorate the
consequences of the genetic lesion. This genetic approach is predicated
on the availability of cloned genes and their flanking sequences, and
molecular clones encoding ODC have been isolated and
characterized from both T. brucei (13) and L. donovani (14). To genetically define the role of ODC in polyamine metabolism and to dissect the polyamine pathway in intact parasites, a
null mutant of L. donovani has been created by
double-targeted gene replacement in which both wild type ODC
alleles have been sequentially eliminated. The phenotypic dissection of
the parasites in which the ODC copy number has been
genetically altered has established the essential role of ODC in
polyamine metabolism, reveals significant discrepancies between the
polyamine pathway of the parasite and host cells, has important
implications in understanding the therapeutic relevance of the
polyamine pathway, and supports a general strategy for the creation of
attenuated strains for vaccine development in prophylaxing leishmaniasis.
Materials, Chemicals, and
Reagents--
[COOH-14C]Ornithine (50-60 mCi/mmol) was
obtained from Moravek Biochemicals (Brea, CA), whereas
[14C]spermidine trihydrochloride (113 mCi/mmol) and
[14C]spermine tetrahydrochloride (115 mCi/mmol) were
procured from Amersham Pharmacia Biotech. Diamines were purchased from
Sigma. DFMO was a gift from the Merrell Dow Research Institute
(Cincinnati, OH). The pX63-NEO and pX63-HYG plasmids used in the
parasite transfections and pSNBR used in the subcloning of a cosmid
fragment were generously provided to this laboratory by Dr. Stephen M. Beverley (Washington University, St. Louis, MO). Purified L. donovani ODC was furnished by Dr. Margaret A. Phillips (University
of Texas Southwestern Medical Center, Dallas, TX). All other materials,
chemicals, and reagents used in these experiments have been described
previously (14-16) and were of the highest quality commercially available.
Cell Culture and Preexisting Cell Lines--
L.
donovani promastigotes, the extracellular insect vector form of
the parasite, were grown in DME-L, a completely defined culture medium
especially designed for the cultivation of Leishmania (17).
In specified experiments, cells were propagated in a modified DME-L
medium, DME-L-CS, in which the bovine serum albumin component of DME-L
was replaced with 10% chicken serum. The DI700 cell line is a wild
type clone of the Sudanese 1S strain of L. donovani that was
used for DNA isolation and library construction and as a recipient
strain in all initial transfections. For the purposes of the genetic
manipulations reported in this study, DI700 is denoted
ODC+/+, in which + refers to the wild type allele. Growth
rate determinations in the presence of polyamines and diamines were
carried out as described previously (9, 14).
DNA Manipulations and Library Construction--
Genomic DNA was
isolated from L. donovani promastigotes by standard
protocols (14, 15). Southern blot analysis was performed as described
previously (14, 15). A cosmid library was prepared from DI700 genomic
DNA that was partially digested with Sau3A, and 30-45-kb
fragments were ligated into the BamHI site of the Supercos 1 cosmid vector using protocols described in the brochure from Stratagene
(La Jolla, CA).
Isolation of a Cosmid Clone Containing ODC--
A cosmid
encompassing ODC designated ODC-5L1 was isolated and
purified using the L. donovani ODC as a probe and stringent hybridization and wash conditions as reported previously (14, 15). The
L. donovani ODC was originally isolated from a bacteriophage clone as described previously by this laboratory (14). Restriction mapping of the cosmid was performed as described in the relevant brochure from Stratagene.
Oligonucleotide Primers--
Primers used in the amplification
of the 5'- and 3'-flanking regions of ODC by the polymerase
chain reaction (PCR) are as follows, with their restriction enzyme
sites underlined: 5'F5', 5'-CCCAAGCTTGTCCACGCTGCACAC-3';
5'F3', 5'-GTTGAACTGGCGGGCCGC-3'; 3'F5',
5'-TCCCCCGGGGGATGCACGGCGACG-3'; and 3'F3',
5'-GAAGATCTGAGAGGCACTTTACTC-3'.
Molecular Constructs for the Replacement of ODC Alleles--
To
construct appropriate vectors for the replacement of each wild type
ODC allele, a 0.8-kb fragment (5'F) consisting of 410 bp of
5'- untranslated DNA and 390 bp of the ODC open reading frame was ligated into the HindIII-SalI site, and
a 2.0-kb fragment of 3'-untranslated DNA (3'F) was inserted into the
SmaI-BglII site of both the pX63-NEO (18) and
pX63-HYG (19) vectors (Fig. 1). Both 5'F and 3'F were amplified by PCR
using standard reaction conditions (15 cycles; 94 °C for 60 s,
50 °C for 30 s, 72 °C for 30 s) for the amplification
of DNAs from plasmid or cosmid DNA templates (16). 5'F was generated
from a 15-kb HindIII-EcoRI fragment of ODC-5L1
encompassing ODC that had been subcloned into pSNBR (20),
whereas 3'F was amplified from the 3.9-kb SalI fragment described by Hanson et al. (14). Sense and antisense primers used in the PCR were 5'F5' and 5'F3' for 5'F and 3'F5' and 3'F3' for
3'F, respectively. These PCR products were digested with the appropriate restriction enzymes; 5'F, which has an internal
SalI site, was cleaved with HindIII and
SalI, whereas 3'F was digested with SmaI and
BglII and ligated sequentially into pX63-NEO and pX63-HYG.
The presence of the flanking regions and their orientation in the
knockout vectors were confirmed by nucleotide sequencing (21) and
restriction mapping. The recombinant pX63-NEO and pX63-HYG vectors
containing the ODC flanking regions are designated
pX63-NEO- Transfections--
Parasites were transfected by electroporation
using conditions identical to those described previously (18).
pX63-NEO- ODC assay--
ODC activity was measured radiometrically as
described previously (14).
Western Blotting--
Polyclonal antibody to purified L. donovani ODC was generated in rabbits by conventional methods
(22). Promastigotes were lysed by sonication, and cell supernatants
were prepared by centrifugation at 20,000 × g. 10 µg
of protein from each cell line were fractionated by SDS-polyacrylamide
gel electrophoresis (23), blotted onto nitrocellulose membranes using a
Semi-Dry Electrophoretic Transfer Cell (Bio-Rad), and subjected to
Western blot analysis by standard protocols (22).
Polyamine Pool Determinations--
5.0 × 108
parasites were harvested and extracted for polyamine pool
determinations with 10% trichloroacetic acid as described previously
(24). An internal 1,7-diaminoheptane standard (40 µM) was
then added to the trichloroacetic acid supernatants. The trichloroacetic acid was extracted with ethyl acetate as reported previously (24), and the samples were dried on a Speed Vac
concentrator. Samples were pre-column derivatized with dansyl chloride
using previously reported protocols (25), except that the sample
volumes were 100 µl. 100 µl of 25% proline were added to scavenge
excess dansyl chloride. The derivatized polyamines were recovered with two ethyl acetate extractions, and the organic layers were pooled and
dried. Samples were resuspended in 200 µl of 95% methanol/5% acetic
acid, and polyamines were analyzed by high performance liquid
chromatography on a Beckman system equipped with a C18 reversed phase column (Bio-Rad) as described previously (25). Relative
fluorescence was measured on a Shimadzu RF-535 fluorescence high
performance liquid chromatography monitor at excitation and emission
wavelengths of 365 and 485 nm, respectively. Peak areas were calculated
using a Hewlett Packard HP3396 series II integrator and compared with
those of known polyamine standards.
Thiol Pool Measurements--
1.0 × 108 cells
were prepared for thiol pool determinations and derivatized with
monobromobimane as described previously (24). Derivatized thiols were
fractionated by high performance liquid chromatography over a Vydac
C18 reversed phase column as reported previously (26).
Relative fluorescence was measured at excitation and emission
wavelengths of 395 and 480 nm, respectively. Peak areas were calculated
as described for the polyamine pool determinations.
Radiolabeled Polyamine Incorporation Experiments--
5 × 106 wild type promastigotes were incubated with 2 µCi of
[14C]spermidine (113 mCi/mmol) or
[14C]spermine (115 mCi/mmol) in 5 ml of DME-L-CS under
normal growth conditions for 48 h. Polyamine pools were processed
and chromatographed as described above. 1-ml fractions were collected,
and radioactivity was quantified by liquid scintillation spectrometry.
The positions of the radiolabeled polyamines were determined by
co-injection with polyamine standards.
Replacement of the ODC Alleles--
To disrupt the ODC
locus, a single copy gene in L. donovani (14), each allele
was sequentially replaced with a drug resistance cassette. The first
ODC allele was displaced with pX63-NEO-
Southern blot analysis of the ODC+/+,
ODC+/n, and ODCn/h strains revealed
the new alleles that had been constituted by homologous gene replacement events (Fig. 2). These altered alleles could be effectively discriminated from the wild type allele by the positions of distinct SacI sites located within the endogenous ODC
locus, pX63-NEO- ODC Expression in ODC+/+, ODC+/n, and
ODCn/h L. donovani--
To evaluate the phenotypic
consequences of ODC replacement, ODC activity and protein
were measured in wild type and genetically manipulated strains. As
shown in Fig. 3, levels of ODC activity in ODC+/+, ODC+/n, and ODCn/h cells
were directly proportional to the ODC copy number. ODC activity in the heterozygous deletion mutant was ~50% that of wild
type cells, and no ODC activity could be detected in the ODCn/h double knockout. Western blot analysis of
ODC+/+, ODC+/n, and ODCn/h cell
extracts demonstrated that the observed reduction in ODC activity in
the single and double knockouts was consistent with diminished cellular
expression of ODC protein (Fig. 4).
Nutritional Requirements of ODCn/h Parasites--
The
nutritional requirements of the
The cellular polyamine requirement in ODCn/h cells could
also be satisfied by the provision of 200 µM spermidine,
although supplementation of the medium with equimolar spermine, a
concentration that did not affect the growth of ODC+/+
parasites, did not support the growth of the knockout (Fig.
6). The experiments establishing whether
spermidine and spermine could overcome cellular ODC deficiency were
conducted in DME-L-CS medium to avoid the polyamine toxicity
attributable to the presence of minute amounts of polyamine oxidase
activity in DME-L medium. Interestingly, the polyamine requirement of
ODCn/h cells could also be fulfilled by supplementing
DME-L-CS with either 1,3-diaminopropane or 1,5-diaminopentane
(cadaverine) (Fig. 6). However, the growth rate of
Polyamine and Thiol Pool Measurements--
The metabolic
consequences of ODC deficiency on polyamine and thiol pools
were also evaluated in
The removal of
Polyamines and their conjugates were also measured in
ODCn/h cells that had been propagated for >3 weeks in
putrescine-deficient DME-L-CS supplemented with spermidine. Under these
conditions, putrescine concentrations in the Polyamine Catabolism in L. donovani--
The failure of spermidine
supplementation to augment cellular putrescine pools of
The creation of a Genetic studies have also demonstrated that ODC is indispensable for
long-term proliferation and viability of T. brucei (33). After replacement of both ODC alleles with drug resistance
cassettes, ODC-deficient T. brucei incubated without
polyamines growth arrested at the G1-S-phase transition of
the cell cycle. Interestingly, a small percentage of the arrested
Although the present results establish a strict requirement for
polyamines in L. donovani, the precise mechanism by which ODC deficiency triggers lethality has not been definitively
established. Removal of Circumvention of the conditionally lethal The polyamine requirement of The ability to create polyamine auxotrophs of Leishmania by
deletion of the ODC locus suggests that ODC could be
targeted by live parasite vaccination strategies against leishmaniasis. A similar vaccine-based approach has been inaugurated using thymidine auxotrophs of L. major in which the dihydrofolate
reductase-thymidylate synthase locus has been replaced. These
dihydrofolate reductase-thymidylate synthase null mutants induce
protective immunity in mice and do not precipitate disease (42). We are
currently evaluating the ability of our odc
cells in polyamine-deficient medium resulted in a rapid and profound
depletion of cellular putrescine pools, although levels of spermidine
were relatively unaffected. Concentrations of trypanothione, a
spermidine conjugate, were also reduced, whereas glutathione
concentrations were augmented. The
odc L. donovani
exhibited an auxotrophy for polyamines that could be circumvented by
the addition of the naturally occurring polyamines, putrescine or
spermidine, to the culture medium. Whereas putrescine supplementation
restored intracellular pools of both putrescine and spermidine,
exogenous spermidine was not converted back to putrescine, indicating
that spermidine alone is sufficient to meet the polyamine requirement,
and that L. donovani does not express the enzymatic
machinery for polyamine degradation. The lack of a polyamine catabolic
pathway in intact parasites was confirmed radiometrically. In addition,
the
odc strain could grow in medium supplemented with
either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but
polyamine auxotrophy could not be overcome by other aliphatic diamines
or spermine. These data establish genetically that ODC is
an essential gene in L. donovani, define the polyamine
requirements of the parasite, and reveal the absence of a
polyamine-degradative pathway.
INTRODUCTION
Top
Abstract
Introduction
References
-difluoromethylornithine (DFMO;
eflornithine), an irreversible inhibitor of ODC (5), exhibits notable
efficacy against the central nervous system phase of African sleeping
sickness caused by Trypanosoma brucei gambiense (3, 6). DFMO
is also active against T. b. rhodesiense and T. congolense in murine models and has proven effective against other
genera of protozoan parasites in vivo and in
vitro, including Plasmodia (7), Giardia (8),
and Leishmania (9). DFMO has been shown to induce a lethal
polyamine depletion in both T. brucei (10) and L. donovani (9), the etiologic agent of visceral leishmaniasis, and
toxicity to both species is ameliorated by polyamine addition (3,
9).
MATERIALS AND METHODS
odc and pX63-HYG-
odc, respectively
(Fig. 1, B and C).
odc and pX63-HYG-
odc were
linearized with HindIII and BglII and gel purified before electroporation. In the construction of the
odc strain, the first wild type ODC allele was
replaced with pX63-NEO-
odc to create the
ODC/odc heterozygote (designated ODC+/n),
whereas the second wild type allele was deleted from the heterozygote with pX63-HYG-
odc to create the homozygous
odc knockout null strain (designated ODCn/h).
Electroporated parasites were maintained in liquid medium for 24 h
before plating on a drug-containing semisolid medium. Drug-resistant clones transfected with pX63-NEO-
odc were isolated from
plates containing 20 µg/ml Geneticin (G418), whereas parasites
transfected with pX63-HYG-
odc were selected in 20 µg/ml
G418, 50 µg/ml hygromycin, and 100 µM putrescine.
Colonies isolated after transfection with either
pX63-NEO-
odc or pX63-HYG-
odc were picked
into 1.0 ml of liquid DME-L containing the relevant selective and
indispensable agents, expanded, and analyzed for the appropriate
allelic replacements by Southern blotting. The ODC+/n and
ODCn/h transformants were maintained continually in the
appropriate selective media unless otherwise indicated.
RESULTS
odc to
create the ODC+/n heterozygote, and the second was replaced
with pX63-HYG-
odc to create the null ODCn/h
odc mutant. In each round of transfection, 4 × 107 promastigotes were transfected with the appropriate
linearized DNA fragments, and ~100 drug-resistant colonies were
obtained from each plating. The homozygotes were selected in medium
supplemented with 100 µM putrescine, because it has been
established previously that pharmacologic simulation of a genetic
deficiency of ODC in L. donovani by the incubation of intact
parasites with DFMO could be circumvented by the addition of the
diamine to the culture medium (9). G418 was also added to the
hygromycin resistance selections to ensure that the second round of
gene targeting yielded cell lines in which pX63-HYG-
odc
had replaced the wild type allele of the ODC+/n heterozygote.
odc, and pX63-HYG-
odc (Fig.
1). These allelic differences are
demonstrated in Fig. 2. Digestion of
genomic DNA prepared from ODC+/+, ODC+/n, and
ODCn/h cells with SacI and hybridization with
the 0.8-kb 5'-flanking probe 5'F (Fig. 1, Probe A) revealed
only the expected wild type hybridization signals at 1.6 and 1.3 kb
(Fig. 2A). A novel 3.2-kb band was observed in
SacI-digested genomic DNA from ODC+/n with a
concomitant diminution of the hybridization intensity of the 1.6-kb
signal from the 5'-flanking region of the wild type allele. A similar
digestion of ODCn/h DNA showed the loss of the 1.6-kb
fragment from the wild type allele and an increase in the hybridization
signal at 3.2 kb. As expected from the restriction maps (Fig. 1), no
changes in the 1.3-kb signal were observed (Fig. 2A). A
parallel digestion of genomic DNA with SacI-SalI
and probing with the 2.0-kb 3'-flanking probe 3'F (Fig. 1, Probe
B) also confirmed the presence of the new alleles in
ODC+/n and ODCn/h cells and the disappearance
of the wild type counterparts. The SacI-SalI band
that hybridizes to 3'F is 2.3 kb (see Fig. 1), whereas the fragments
from the alleles disrupted by pX63-NEO-
odc and
pX63-HYG-
odc are 2.8 kb (Fig. 2B). Finally, to
establish that the drug-resistant clones were truly deficient in
ODC coding region sequences, genomic DNA from the three
genotypes was digested with SalI and probed with a 1.3-kb
BamHI-StuI fragment located within the protein
coding portion of ODC (Fig. 1, Probe C). As anticipated, a 3.9-kb hybridization signal corresponding to the wild
type allele was observed in the ODC+/+ line. The same
fragment, exhibiting approximately half the signal intensity of the
wild type 3.9-kb band, was observed in the ODC+/n
heterozygote, whereas the band was absent in the ODCn/h
knockout. It is worth noting that after both rounds of transfection, clones displaying genetic events other than simple gene replacements were detected by Southern blotting at a frequency of ~20%, but these
more complex genetic alterations were not analyzed further.
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Fig. 1.
Restriction maps of the ODC locus
and plasmid constructs used in targeted gene replacement
protocols. Restriction maps of the ODC locus
(A) and the pX63-NEO- odc (B) and
pX63-HYG-
odc (C) knockout vectors are
indicated. The ODC and neomycin and hygromycin
phosphotransferase markers are translated from left to right and are
indicated by white unfilled rectangular boxes. The 0.8-kb
5'F and 2.0-kb 3'F-flanking regions that were amplified by PCR and used
in Southern blotting experiments (see Fig. 2) are indicated by the
thick black lines. Probes A, B, and C
correspond to 5'F, 3'F, and the ODC protein coding region,
respectively, and are indicated by the thick gray lines. The
predicted sizes of the restriction fragments that hybridize to
ODC locus probes and all appropriate restriction sites are
shown.
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Fig. 2.
Southern blot analysis of wild type and
mutant parasites. 10 µg of total genomic DNA from wild type
(ODC+/+), G418-resistant single replacement
(ODC+/n), and G418/hygromcyin-resistant double replacement
(ODCn/h) were digested with SacI (A),
SacI-SalI (B), and SalI
(C), fractionated on agarose gels, and transferred to Nylon
membranes. Nylon membranes were then hybridized to either 5'F
(A), 3'F (B), or a probe to the deleted portions
of the protein coding region of ODC (C). The
positions in the ODC genomic locus to which each of these
probes correspond are indicated in Fig. 1. The size markers in kb are
indicated on the left.
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Fig. 3.
ODC activity in wild type and mutant
cells. ODC activity was measured in extracts of wild type
ODC+/+ ( ), ODC+/n (
), and
ODCn/h (
) cells as described previously (14).
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Fig. 4.
Western blot analysis of expressed ODC
protein in wild type and mutant parasites. Polyclonal antiserum
against L. donovani ODC was generated in rabbits and used to
detect ODC protein in fractionated extracts obtained from exponentially
growing ODC+/+, ODC+/n, and ODCn/h
cells. Molecular mass markers are indicated on the
right.
odc parasites were also
evaluated. As demonstrated in Fig. 5, the
ODCn/h double knockout could not grow in DME-L medium in
the absence of putrescine, whereas ODC+/n grew as
proficiently as the ODC+/+ strain (data not shown). The
addition of 200 µM putrescine to the culture medium
averted the lethal consequences of ODC deficiency, and the
growth rate of ODCn/h cells in the presence of putrescine
was indistinguishable from that of the ODC+/+ and
ODC+/n cell lines (Fig. 5). Putrescine did not affect the
growth rate of either the ODC+/+ or ODC+/n cell
lines. Parallel results were obtained with all three strains cultivated
in DME-L-CS (data not shown). No morphological distinctions were noted
among the three strains by light microscopy as long as
ODCn/h cells were maintained in putrescine-supplemented
medium.
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Fig. 5.
Growth of wild type and mutant parasites in
DME-L-CS medium in the absence or presence of putrescine. The
ability of ODC+/+ ( ) and ODCn/h (
,
)
cells to grow in DME-L-CS in the absence (empty symbols) or presence
(filled symbols) of 200 µM putrescine is indicated. This
experiment is representative of approximately four others, each of
which yielded similar results.
odc cells in DME-L-CS supplemented with either
1,3-diaminopropane or cadaverine was somewhat less than that
in medium supplemented with putrescine, although the final cell
densities were similar (data not shown). The ODCn/h cells
could be propagated continuously in DME-L-CS supplemented with 200 µM concentrations of either putrescine, spermidine,
1,3-diaminopropane, or cadaverine for >6 months. The
odc parasites could not grow in DME-L-CS supplemented
with other diamines, including 1,2-diaminoethane, 1,6-diaminohexane,
1,7-diaminoheptane, 1,8-diaminooctane, 1,10-diaminodecane, and
1,12-diaminododecane, at concentrations that were nontoxic to wild type
parasites.
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Fig. 6.
Growth of odc mutants in
DME-L-CS medium supplemented with various polyamines and diamines.
The ability of ODCn/h cells to grow in DME-L-CS in the
presence of 200 µM concentrations of putrescine (
),
spermidine (
), spermine (
), 1,3-diaminopropane (
), or
cadaverine (
) is compared.
odc cells. Thiol pools were measured, because Leishmania and other trypanosomatid
protozoa contain millimolar concentrations of the spermidine-containing trypanothione molecule (27), a thiol that likely serves as a general
reductant in these parasites (28). As shown in Table I, exponentially growing wild type and
ODC+/n cells contained commensurate concentrations of
putrescine, spermidine, trypanothione, glutathionylspermidine, and
glutathione. The intracellular putrescine and spermidine pools of the
odc cells expanded in putrescine-supplemented medium were
also comparable to those of wild type and ODC+/n cells,
although the thiol concentrations were significantly higher. No
spermine was detected in any of these cell lines, which is consistent
with previous observations that L. donovani lack spermine (9).
Polyamine and thiol levels in wild type and mutant L. donovani
odc cells from putrescine-supplemented
DME-L-CS precipitated a rapid depletion of cellular putrescine pools, although the levels of spermidine remained relatively constant after 12 days of maintenance in medium lacking putrescine (Fig. 7A). Reduction in
ODCn/h cell numbers was not observed until day 7 after
removal of the exogenous putrescine, and some augmentation in the cell
density was observed through the first 4 days of incubation (Fig. 7). This marginal cell proliferation could be attributed to the fact that
Leishmania accommodate sufficient polyamine pools to
maintain viability and enable minimal growth in the absence of both
polyamine biosynthesis and polyamine salvage. However, throughout the
incubation period in unsupplemented DME-L-CS, levels of trypanothione
in
odc cells were much lower than those observed in
ODC+/+, ODC+/n, or
ODCn/h parasites grown in putrescine-containing medium,
although trypanothione levels remained relatively constant, albeit low,
after 24 h in the absence of putrescine (Fig. 7B).
Glutathionylspermidine levels fluctuated slightly in the
odc cell line maintained in medium lacking polyamine,
whereas cellular glutathione concentrations increased fairly
substantially, i.e. ~2-fold.
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Fig. 7.
Polyamine and thiol pools in
ODCn/h cells incubated in medium lacking polyamine.
ODCn/h cells were removed from DME-L supplemented with 200 µM putrescine and resuspended in fresh DME-L lacking
putrescine as described under "Materials and Methods." At the time
of resuspension and at 24-h intervals thereafter, 5 × 108
cells were removed for putrescine ( ) and spermidine (
)
measurements (A), and 108 cells were removed for
the determination of trypanothione (
), glutathionylspermidine (
),
and glutathione (
) pools (B). Cell density was monitored
by Coulter counting (
).
odc strain
were 4% of those of wild type parasites, whereas spermidine and
trypanothione pools were comparable. Polyamine and thiol pools were
also measured in
odc parasites grown in either
1,3-diaminopropane or cadaverine (Table I). Both diamines were
accumulated by the ODCn/h cells, and a concomitant
depletion of naturally occurring polyamines was observed. Cellular
putrescine pools were negligible, and spermidine pools were markedly
depleted in
odc cells maintained in either 1,3-diaminopropane or cadaverine compared with the knockout parasites grown in putrescine or wild type parasites. The null mutant grown in
either spermidine, spermine, or cadaverine also appeared to accumulate
small amounts of 1,3-diaminopropane. The origin of this anomaly could
be imputed to contaminants in the spermidine, spermine, and cadaverine
additives. Trypanothione pools in ODCn/h cells grown in
DME-L-CS supplemented with either 1,3-diaminopropane or cadaverine were
also very low (2.0 and 10.5% of the levels found in
putrescine-supplemented
odc parasites; Table I).
odc parasites suggested that L. donovani lack
a polyamine-degradative pathway. To verify this conjecture, wild type
parasites were incubated with either [14C]spermidine or
[14C]spermine to determine whether the polyamines could
be catabolized. As demonstrated in Fig.
8, L. donovani do not convert
extracellular spermidine or spermine to putrescine, although each
radiolabel is accumulated by the parasites in unaltered form.
Intracellular [14C]spermidine is observed in
promastigotes that had been incubated with [14C]spermine,
but this could be ascribed to a trace [14C]spermidine
contaminant present in the radiolabeled spermine (data not shown).
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Fig. 8.
Polyamine catabolism in L. donovani. ODC+/+ L. donovani were
incubated with [14C]spermidine (A) or
[14C]spermine (B) as reported under
"Materials and Methods." Cells were harvested and washed, and
polyamines were fractionated as described. 1.0-ml fractions were then
counted by liquid scintillation. The migration of known standards of
putrescine (PUT), spermidine (SPD), and spermine
(SPN) is indicated.
DISCUSSION
odc strain of
Leishmania by double-targeted gene replacement establishes
that ODC plays an essential housekeeping function in this
genus of protozoan parasite. Using independent drug resistance
cassettes, ODC coding sequences were expunged from wild type
L. donovani in two discrete steps, and the presumptive heterozygote generated after the first round of transfection and selection appeared to contain only one intact ODC allele as
measured by loss of the hybridization signal and a ~50% reduction of
ODC activity and protein as compared with the ODC+/+
parent. These data confirmed previous results obtained by Southern blotting that L. donovani is diploid at the ODC
locus, and that ODC is a single copy gene (14). The
observation that
odc cells cannot survive without
putrescine supplementation of the culture medium demonstrates
genetically that ODC and polyamines are indispensable to L. donovani and underscores the potential of the polyamine pathway as
a therapeutic target. The requirement for ODC is consistent with
previous observations that DFMO toxicity in L. donovani can be bypassed by the addition of putrescine to the culture medium (9).
Furthermore, the growth phenotype of the
odc strain
authenticates that ODC is the sole enzyme that initiates polyamine
biosynthesis in L. donovani. This conclusion is an important
distinction, because Escherichia coli and plants can
synthesize putrescine from arginine via an arginine
decarboxylase-agmatine ureohydrolase pathway (2). Moreover, there have
been reports (29, 30), although unconfirmed (31, 32), that T. cruzi, a protozoan parasite related to Leishmania, expresses an arginine decarboxylase activity that can be targeted by
specific inhibitors.
odc population remained viable 7-8 weeks after
putrescine withdrawal (33). ODC-deficient mutants of mammalian cells
(34) and Neurospora crassa (35) also could not thrive in the
absence of polyamine supplement, confirming that ODC activity is
mandatory for the growth of these cells. However, ODC is not essential
for E. coli (36), which has an alternative pathway for
polyamine synthesis, or for some mutant strains of Saccharomyces
cerevisiae that are both ODC and polyamine deficient (37).
Moreover, recent studies have strongly implied that T. cruzi
lack a polyamine biosynthetic pathway altogether and are therefore
obligate scavengers of polyamines (31, 32).
odc cells from
putrescine-supplemented medium triggered a rapid and virtually complete
depletion of cellular putrescine pools without a concomitant diminution
in spermidine levels. A similar obliteration of putrescine pools is
also observed when wild type L. donovani are treated with
DFMO (9). Incubation of ODCn/h parasites in the absence of
exogenous polyamines also prompted a rapid decrease in the cellular
levels of trypanothione, which were subsequently maintained at a low
level, whereas concentrations of glutathione increased steadily
throughout the prolonged incubation. The depletion of trypanothione
could be ascribed either to the reduced flux through the polyamine
biosynthetic pathway or to trypanothione turnover to replenish
spermidine pools. It is possible that this augmentation of glutathione
is a cellular compensation mechanism to preserve the reductive
potential of the intracellular environment when trypanothione pools are
reduced. The trypanothione depletion and glutathione elevation in
putrescine-depleted ODCn/h L. donovani parallels
results obtained with T. brucei in which glutathionylspermidine and trypanothione levels were both substantially depleted and glutathione levels were augmented after DFMO treatment (26). Thus, cessation of parasite growth in polyamine-deficient medium
correlates with depletion of both the putrescine and trypanothione pools.
odc mutation
can be achieved by the addition of either putrescine or spermidine to
the culture medium. Given that the null mutant propagated in spermidine-supplemented medium exhibited a profound reduction of the
putrescine pool, the most obvious inference is that L. donovani does not require intracellular putrescine to survive, and
that spermidine is both sufficient and necessary to satisfy the
polyamine requirement of the parasite. Spermine, a major polyamine of
mammalian cells, is not detected in L. donovani (9),
supporting the lack of a spermine synthase activity. Moreover,
exogenous spermine does not satisfy the polyamine requirement of the
odc promastigotes, a distinction from what is observed in
mammalian cells in which exogenous spermine can circumvent a genetic
deficiency in ODC activity (34). In mammalian cells, spermine is
converted to spermidine and spermidine is converted to putrescine by an identical two-enzyme pathway (38) consisting of spermidine/spermine N1-acetyltransferase and polyamine oxidase. The inability
of
odc cells to use spermine as a polyamine source or to
replenish their putrescine pools from extracellular spermidine implied
that L. donovani lacks the spermidine/spermine
N1-acetyltransferase/polyamine oxidase pathway altogether.
This was then confirmed radiometrically using both
[14C]spermidine and [14C]spermine.
Considering that parasitic nematodes (39) and E. coli (40)
can acetylate naturally occurring polyamines, the lack of a counterpart
pathway in L. donovani is unusual. Thus, it seems that the
equilibrium between the putrescine and spermidine pools in L. donovani is governed exclusively by spermidine synthase activity,
unlike mammalian cells, in which intracellular polyamines are regulated
by a delicate balance among the anabolic enzymes, spermidine and
spermine synthase, and the catabolic enzymes, spermidine/spermine N1-acetyltransferase and polyamine oxidase. Although the
polyamine aminopropyltransferases have not been generally embraced as
therapeutic targets, the limited complement of polyamine enzymes in
L. donovani provides a rational basis for the utilization of
spermidine synthase inhibitors (41) in conjunction with exogenous
spermidine/spermine in therapies.
odc cells can also be
fulfilled by either 1,3-diaminopropane or cadaverine, because the
polyamine auxotrophs can grow continuously in DME-L-CS supplemented
with either diamine, and each is taken up and accumulated by the
parasites. Essentially no putrescine was detected in the knockout cells
incubated with either diamine, which supports the contention that
putrescine is not an essential metabolite for L. donovani.
Small quantities of spermidine, however, were observed in the
1,3-diaminopropane- and cadaverine-propagated cultures. The source of
this spermidine is unclear, although it may be present in the
supplemented culture medium in insufficient amounts to support the
growth of the
odc cells. Glutathionylspermidine and
trypanothione levels were also markedly reduced compared with either
wild type parasites or
odc cells propagated in
putrescine-supplemented medium. Although previous investigators
detected significant amounts of homotrypanothione, the
cadaverine-containing analog of trypanothione, in T. cruzi (31), we were unable to distinguish homotrypanothione and trypanothione in our high performance liquid chromatography system. These data imply
that these thiols are not essential for the continual propagation of
L. donovani promastigotes, at least in the absence of
environmental insult.
odc to infect and
proliferate within human macrophages, the cell type in which the human
stage of the parasite resides.
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FOOTNOTES |
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* This work was supported in part by Grant AI 41622 from the National Institute of Allergy and Infectious Disease 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of an N. L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon.
A Burroughs Wellcome Fund Scholar in Molecular Parasitology.
** To whom all correspondence should be addressed. Tel.: 503-494-8437; Fax: 503-494-8393; E-mail: ullmanb{at}ohsu.edu.
The abbreviations used are:
ODC, ornithine
decarboxylase; DFMO, -difluoromethylornithine; DME-L, Dulbecco's
modified Eagle-Leishmania medium; DME-L-CS, DME-L plus 10%
chicken serum; kb, kilobase pairs; PCR, polymerase chain reaction.
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REFERENCES |
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