![Polyamine catabolism in L. donovani. ODC / L. donovani were incubated with [ 14 C]spermidine (A) or [ 14 C]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.](https://www.researchgate.net/profile/Alan-Fairlamb/publication/13368122/figure/fig3/AS:601774020902913@1520485515071/Polyamine-catabolism-in-L-donovani-ODC-L-donovani-were-incubated-with-14_Q320.jpg)
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Ornithine Decarboxylase Gene Deletion Mutants of
Leishmania donovani*
(Received for publication, September 25, 1998, and in revised form, November 13, 1998)
Yuqui Jiang‡§, Sigrid C. Roberts‡, Armando Jardim‡, Nicola S. Carter‡, Sarah Shih‡,
Mark Ariyanayagam
¶
, Alan H. Fairlamb
¶
, and Buddy Ullman‡
i
**
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
A knockout strain of Leishmania donovani lacking
both ornithine decarboxylase (ODC) alleles has been
created by targeted gene replacement. Growth of Dodc
cells in polyamine-deficient medium resulted in a rapid
and profound depletion of cellular putrescine pools, al-
though levels of spermidine were relatively unaffected.
Concentrations of trypanothione, a spermidine conju-
gate, were also reduced, whereas glutathione concentra-
tions were augmented. The Dodc L. donovani exhibited
an auxotrophy for polyamines that could be circum-
vented by the addition of the naturally occurring poly-
amines, putrescine or spermidine, to the culture me-
dium. Whereas putrescine supplementation restored
intracellular pools of both putrescine and spermidine,
exogenous spermidine was not converted back to pu-
trescine, indicating that spermidine alone is sufficient
to meet the polyamine requirement, and that L. dono-
vani does not express the enzymatic machinery for poly-
amine degradation. The lack of a polyamine catabolic
pathway in intact parasites was confirmed radiometri-
cally. In addition, the Dodc strain could grow in medium
supplemented with either 1,3-diaminopropane or 1,5-
diaminopentane (cadaverine), but polyamine auxotro-
phy 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.
Polyamines are cationic compounds that play essential roles
in cell proliferation, differentiation, and macromolecular syn-
thesis (1–3). Ornithine decarboxylase (ODC)
1
catalyzes the con-
version 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-
a
-difluoromethylor-
nithine (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).
The ability of trypanosomatids to undergo a very high fre-
quency 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 sur-
vival 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 avail-
ability of cloned genes and their flanking sequences, and mo-
lecular clones encoding ODC have been isolated and character-
ized 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 impor-
tant 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 AND METHODS
Materials, Chemicals, and Reagents—[COOH-
14
C]Ornithine (50–60
mCi/mmol) was obtained from Moravek Biochemicals (Brea, CA),
whereas [
14
C]spermidine trihydrochloride (113 mCi/mmol) and
[
14
C]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 Southwest-
ern 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%
* 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. This 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.
i
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@ohsu.edu.
1
The abbreviations used are: ODC, ornithine decarboxylase; DFMO,
a
-difluoromethylornithine; DME-L, Dulbecco’s modified Eagle-Leish-
mania medium; DME-L-CS, DME-L plus 10% chicken serum; kb, kilo-
base pairs; PCR, polymerase chain reaction.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 6, Issue of February 5, pp. 3781–3788, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 3781
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
1/1
, in which 1 refers to the wild type allele. Growth rate
determinations in the presence of polyamines and diamines were car-
ried 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 encompass-
ing ODC designated ODC-5L1 was isolated and purified using the L.
donovani ODC as a probe and stringent hybridization and wash condi-
tions as reported previously (14, 15). The L. donovani ODC was origi-
nally 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 59-
and 39-flanking regions of ODC by the polymerase chain reaction (PCR)
are as follows, with their restriction enzyme sites underlined: 59F59,
59-CCCAAGCTTGTCCACGCTGCACAC-39;59F39,59-GTTGAACTGGC-
GGGCCGC-39;39F59,59-TCCCCCGGGGGATGCACGGCGACG-39; and
39F39,59-GAAGATCTGAGAGGCACTTTACTC-39.
Molecular Constructs for the Replacement of ODC Alleles—To con-
struct appropriate vectors for the replacement of each wild type ODC
allele, a 0.8-kb fragment (59F) consisting of 410 bp of 59- 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 39-untranslated DNA (39F)
was inserted into the SmaI-BglII site of both the pX63-NEO (18) and
pX63-HYG (19) vectors (Fig. 1). Both 59F and 39F 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). 59F was generated from a 15-kb HindIII-
EcoRI fragment of ODC-5L1 encompassing ODC that had been sub-
cloned into pSNBR (20), whereas 39F was amplified from the 3.9-kb SalI
fragment described by Hanson et al. (14). Sense and antisense primers
used in the PCR were 59F59 and 59F39 for 59F and 39F59 and 39F39 for
39F, respectively. These PCR products were digested with the appropri-
ate restriction enzymes; 59F, which has an internal SalI site, was
cleaved with HindIII and SalI, whereas 39F 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 contain-
ing the ODC flanking regions are designated pX63-NEO-Dodc and
pX63-HYG-Dodc, respectively (Fig. 1, B and C).
Transfections—Parasites were transfected by electroporation using
conditions identical to those described previously (18). pX63-NEO-Dodc
and pX63-HYG-Dodc were linearized with HindIII and BglII and gel
purified before electroporation. In the construction of the Dodc strain,
the first wild type ODC allele was replaced with pX63-NEO-Dodc to
create the ODC/odc heterozygote (designated ODC
1/n
), whereas the
second wild type allele was deleted from the heterozygote with pX63-
HYG-Dodc to create the homozygous Dodc knockout null strain (desig-
nated ODC
n/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-Dodc were isolated
from plates containing 20
m
g/ml Geneticin (G418), whereas parasites
transfected with pX63-HYG-Dodc were selected in 20
m
g/ml G418, 50
m
g/ml hygromycin, and 100
m
M putrescine. Colonies isolated after
transfection with either pX63-NEO-Dodc or pX63-HYG-Dodc were
picked into 1.0 ml of liquid DME-L containing the relevant selective and
indispensable agents, expanded, and analyzed for the appropriate al-
lelic replacements by Southern blotting. The ODC
1/n
and ODC
n/h
trans-
formants were maintained continually in the appropriate selective me-
dia unless otherwise indicated.
ODC assay—ODC activity was measured radiometrically as de-
scribed 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 cen-
trifugation at 20,000 3 g.10
m
g 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 3 10
8
parasites were harvested
and extracted for polyamine pool determinations with 10% trichloro-
acetic acid as described previously (24). An internal 1,7-diaminohep-
tane standard (40
m
M) was then added to the trichloroacetic acid super-
natants. 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 chlo-
ride using previously reported protocols (25), except that the sample
volumes were 100
m
l. 100
m
l 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
m
l of 95% methanol/5% acetic
acid, and polyamines were analyzed by high performance liquid chro-
matography on a Beckman system equipped with a C
18
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 3 10
8
cells were prepared for thiol
pool determinations and derivatized with monobromobimane as de-
scribed previously (24). Derivatized thiols were fractionated by high
performance liquid chromatography over a Vydac C
18
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 3 10
6
wild
type promastigotes were incubated with 2
m
Ci of [
14
C]spermidine (113
mCi/mmol) or [
14
C]spermine (115 mCi/mmol) in 5 ml of DME-L-CS
under normal growth conditions for 48 h. Polyamine pools were pro-
cessed and chromatographed as described above. 1-ml fractions were
collected, and radioactivity was quantified by liquid scintillation spec-
trometry. The positions of the radiolabeled polyamines were deter-
mined by co-injection with polyamine standards.
RESULTS
Replacement of the ODC Alleles—To disrupt the ODC locus,
a single copy gene in L. donovani (14), each allele was sequen-
tially replaced with a drug resistance cassette. The first ODC
allele was displaced with pX63-NEO-Dodc to create the ODC
1/n
heterozygote, and the second was replaced with pX63-HYG-
Dodc to create the null ODC
n/h
Dodc mutant. In each round of
transfection, 4 3 10
7
promastigotes were transfected with the
appropriate linearized DNA fragments, and ;100 drug-resis-
tant colonies were obtained from each plating. The homozy-
gotes were selected in medium supplemented with 100
m
M
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-Dodc had re-
placed the wild type allele of the ODC
1/n
heterozygote.
Southern blot analysis of the ODC
1/1
, ODC
1/n
, and ODC
n/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-Dodc, and pX63-HYG-Dodc
(Fig. 1). These allelic differences are demonstrated in Fig. 2.
Digestion of genomic DNA prepared from ODC
1/1
, ODC
1/n
,
and ODC
n/h
cells with SacI and hybridization with the 0.8-kb
59-flanking probe 59F (Fig. 1, Probe A) revealed only the ex-
pected 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
1/n
with a concomitant diminution of
the hybridization intensity of the 1.6-kb signal from the 59-
ODC Deficiency in L. donovani3782
flanking region of the wild type allele. A similar digestion of
ODC
n/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 39-flanking probe 39F (Fig. 1, Probe B) also confirmed the
presence of the new alleles in ODC
1/n
and ODC
n/h
cells and the
disappearance of the wild type counterparts. The SacI-SalI
band that hybridizes to 39F is 2.3 kb (see Fig. 1), whereas the
fragments from the alleles disrupted by pX63-NEO-Dodc and
pX63-HYG-Dodc 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 frag-
ment located within the protein coding portion of ODC (Fig. 1,
Probe C). As anticipated, a 3.9-kb hybridization signal corre-
sponding to the wild type allele was observed in the ODC
1/1
line. The same fragment, exhibiting approximately half the
signal intensity of the wild type 3.9-kb band, was observed in
the ODC
1/n
heterozygote, whereas the band was absent in the
ODC
n/h
knockout. It is worth noting that after both rounds of
transfection, clones displaying genetic events other than sim-
ple gene replacements were detected by Southern blotting at a
frequency of ;20%, but these more complex genetic alterations
were not analyzed further.
ODC Expression in ODC
1/1
, ODC
1/n
, and ODC
n/h
L. dono-
vani—To evaluate the phenotypic consequences of ODC re-
placement, ODC activity and protein were measured in wild
type and genetically manipulated strains. As shown in Fig. 3,
FIG.2. Southern blot analysis of
wild type and mutant parasites. 10
m
g
of total genomic DNA from wild type
(ODC
1/1
), G418-resistant single replace-
ment (ODC
1/n
), and G418/hygromcyin-re-
sistant double replacement (ODC
n/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 ei-
ther 59F(A), 39F(B), or a probe to the
deleted portions of the protein coding re-
gion 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.
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-Dodc (B) and pX63-HYG-
Dodc (C) knockout vectors are indicated.
The ODC and neomycin and hygromycin
phosphotransferase markers are trans-
lated from left to right and are indicated
by white unfilled rectangular boxes. The
0.8-kb 59F and 2.0-kb 39F-flanking re-
gions 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 59F, 39F, 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.
ODC Deficiency in L. donovani 3783
levels of ODC activity in ODC
1/1
, ODC
1/n
, and ODC
n/h
cells
were directly proportional to the ODC copy number. ODC ac-
tivity in the heterozygous deletion mutant was ;50% that of
wild type cells, and no ODC activity could be detected in the
ODC
n/h
double knockout. Western blot analysis of ODC
1/1
,
ODC
1/n
, and ODC
n/h
cell extracts demonstrated that the ob-
served reduction in ODC activity in the single and double
knockouts was consistent with diminished cellular expression
of ODC protein (Fig. 4).
Nutritional Requirements of ODC
n/h
Parasites—The nutri-
tional requirements of the Dodc parasites were also evaluated.
As demonstrated in Fig. 5, the ODC
n/h
double knockout could
not grow in DME-L medium in the absence of putrescine,
whereas ODC
1/n
grew as proficiently as the ODC
1/1
strain
(data not shown). The addition of 200
m
M putrescine to the
culture medium averted the lethal consequences of ODC defi-
ciency, and the growth rate of ODC
n/h
cells in the presence of
putrescine was indistinguishable from that of the ODC
1/1
and
ODC
1/n
cell lines (Fig. 5). Putrescine did not affect the growth
rate of either the ODC
1/1
or ODC
1/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 ODC
n/h
cells were maintained in putrescine-supplemented medium.
The cellular polyamine requirement in ODC
n/h
cells could
also be satisfied by the provision of 200
m
M spermidine, al-
though supplementation of the medium with equimolar sperm-
ine, a concentration that did not affect the growth of ODC
1/1
parasites, did not support the growth of the knockout (Fig. 6).
The experiments establishing whether spermidine and sperm-
ine could overcome cellular ODC deficiency were conducted in
DME-L-CS medium to avoid the polyamine toxicity attributa-
ble to the presence of minute amounts of polyamine oxidase
activity in DME-L medium. Interestingly, the polyamine re-
quirement of ODC
n/h
cells could also be fulfilled by supplement-
ing DME-L-CS with either 1,3-diaminopropane or 1,5-diamino-
pentane (cadaverine) (Fig. 6). However, the growth rate of Dodc
cells in DME-L-CS supplemented with either 1,3-diamino-
propane or cadaverine was somewhat less than that in medium
supplemented with putrescine, although the final cell densities
were similar (data not shown). The ODC
n/h
cells could be prop-
agated continuously in DME-L-CS supplemented with 200
m
M
concentrations of either putrescine, spermidine, 1,3-diamino-
propane, or cadaverine for .6 months. The Dodc parasites
could not grow in DME-L-CS supplemented with other dia-
mines, 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.
Polyamine and Thiol Pool Measurements—The metabolic
consequences of ODC deficiency on polyamine and thiol pools
were also evaluated in Dodc cells. Thiol pools were measured,
because Leishmania and other trypanosomatid protozoa con-
tain 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
1/n
cells contained
commensurate concentrations of putrescine, spermidine,
trypanothione, glutathionylspermidine, and glutathione. The
FIG.3.ODC activity in wild type and mutant cells. ODC activity
was measured in extracts of wild type ODC
1/1
(●), ODC
1/n
(M), and
ODC
n/h
(Œ) cells as described previously (14).
FIG.4.Western blot analysis of expressed ODC protein in wild
type and mutant parasites. Polyclonal antiserum against L. dono-
vani ODC was generated in rabbits and used to detect ODC protein in
fractionated extracts obtained from exponentially growing ODC
1/1
,
ODC
1/n
, and ODC
n/h
cells. Molecular mass markers are indicated on the
right.
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
1/1
(●) and ODC
n/h
(f, M) cells to grow in DME-L-CS in the
absence (empty symbols) or presence (filled symbols) of 200
m
M putres-
cine is indicated. This experiment is representative of approximately
four others, each of which yielded similar results.
ODC Deficiency in L. donovani3784
intracellular putrescine and spermidine pools of the Dodc cells
expanded in putrescine-supplemented medium were also com-
parable to those of wild type and ODC
1/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).
The removal of Dodc 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 pu-
trescine (Fig. 7A). Reduction in ODC
n/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 Leishma-
nia accommodate sufficient polyamine pools to maintain via-
bility and enable minimal growth in the absence of both poly-
amine biosynthesis and polyamine salvage. However,
throughout the incubation period in unsupplemented DME-L-
CS, levels of trypanothione in Dodc cells were much lower than
those observed in ODC
1/1
, ODC
1/n
, or ODC
n/h
parasites grown
in putrescine-containing medium, although trypanothione lev-
els remained relatively constant, albeit low, after 24 h in the
absence of putrescine (Fig. 7B). Glutathionylspermidine levels
fluctuated slightly in the Dodc cell line maintained in medium
lacking polyamine, whereas cellular glutathione concentra-
tions increased fairly substantially, i.e. ;2-fold.
Polyamines and their conjugates were also measured in
ODC
n/h
cells that had been propagated for .3 weeks in pu-
trescine-deficient DME-L-CS supplemented with spermidine.
Under these conditions, putrescine concentrations in the Dodc
strain were 4% of those of wild type parasites, whereas sper-
midine and trypanothione pools were comparable. Polyamine
and thiol pools were also measured in Dodc parasites grown in
either 1,3-diaminopropane or cadaverine (Table I). Both dia-
mines were accumulated by the ODC
n/h
cells, and a concomi-
tant depletion of naturally occurring polyamines was observed.
Cellular putrescine pools were negligible, and spermidine pools
were markedly depleted in Dodc 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-diaminopro-
pane. The origin of this anomaly could be imputed to contam-
inants in the spermidine, spermine, and cadaverine additives.
Trypanothione pools in ODC
n/h
cells grown in DME-L-CS sup-
plemented with either 1,3-diaminopropane or cadaverine were
also very low (2.0 and 10.5% of the levels found in putrescine-
supplemented Dodc parasites; Table I).
Polyamine Catabolism in L. donovani—The failure of sper-
midine supplementation to augment cellular putrescine pools
of Dodc parasites suggested that L. donovani lack a polyamine-
degradative pathway. To verify this conjecture, wild type par-
asites were incubated with either [
14
C]spermidine or [
14
C]sper-
mine 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 [
14
C]spermidine is observed in
promastigotes that had been incubated with [
14
C]spermine, but
this could be ascribed to a trace [
14
C]spermidine contaminant
present in the radiolabeled spermine (data not shown).
DISCUSSION
The creation of a Dodc strain of Leishmania by double-tar-
geted gene replacement establishes that ODC plays an essen-
tial housekeeping function in this genus of protozoan parasite.
Using independent drug resistance cassettes, ODC coding se-
quences 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
1/1
parent. These data con-
firmed 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 Dodc cells cannot survive
without putrescine supplementation of the culture medium
demonstrates genetically that ODC and polyamines are indis-
pensable to L. donovani and underscores the potential of the
FIG.6.Growth of Dodc mutants in DME-L-CS medium supple-
mented with various polyamines and diamines. The ability of
ODC
n/h
cells to grow in DME-L-CS in the presence of 200
m
M concen-
trations of putrescine (●), spermidine (f), spermine (‚), 1,3-diamino-
propane (E), or cadaverine (M) is compared.
T
ABLE I
Polyamine and thiol levels in wild type and mutant L. donovani
Polyamine and thiol levels (nmol/10
8
cells) were determined as described under “Materials and Methods” for wild type (ODC
1/1
), heterozygous
(ODC
1/h
), and homozygous (ODC
n/h
). These pools were measured in exponentially growing cells in DME-L-CS in triplicate with the indicated
additions. GSH, glutathione; GS-SPD, glutathionylspermidine; T(SH)2, trypanothione; PUT, putrescine; SPD, spermidine; CAD, cadaverine; 1,3
DP (1,3-diaminopropane); SPM, spermine; ND, not detected.
Cell line Additions GSH GS-SPD T(SH)2 PUT SPD CAD 1,3 DP SPM
ODC
1/1
None 2.15 6 0.37 0.33 6 0.07 8.23 6 1.08 5.00 6 0.06 2.60 6 0.06 ND ND ND
ODC
1/h
None 3.31 6 0.92 0.17 6 0.08 5.31 6 0.43 4.92 6 0.44 2.80 6 0.20 ND ND ND
ODC
n/h
1,3-Diaminopropane 4.41 6 1.00 0.01 6 0.01 0.26 6 0.08 0.06 6 0.04 0.30 6 0.04 ND 19.80 6 0.24 ND
ODC
n/h
Putrescine 9.81 6 1.80 1.23 6 0.41 12.86 6 2.75 3.07 6 0.74 3.14 6 0.34 ND ND ND
ODC
n/h
Cadaverine 2.87 6 0.22 0.05 6 0.03 1.36 6 0.23 0.04 6 0.02 0.40 6 0.06 4.66 6 0.48 0.11 6 0.06 ND
ODC
n/h
Spermidine 6.91 6 1.23 0.54 6 0.10 7.04 6 0.79 0.19 6 0.08 3.02 6 0.32 ND 0.46 6 0.03 ND
ODC
n/h
Spermine 7.50 6 3.10 0.33 6 0.21 3.09 6 0.96 0.76 6 0.02 2.42 6 0.16 ND 1.60 6 0.10 0.36 6 0.02
ODC Deficiency in L. donovani 3785
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 Dodc strain authenticates that ODC is the sole
enzyme that initiates polyamine biosynthesis in L. donovani.
This conclusion is an important distinction, because Esche-
richia coli and plants can synthesize putrescine from arginine
via an arginine decarboxylase-agmatine ureohydrolase path-
way (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.
Genetic studies have also demonstrated that ODC is indis-
pensable for long-term proliferation and viability of T. brucei
(33). After replacement of both ODC alleles with drug resist-
ance cassettes, ODC-deficient T. brucei incubated without poly-
amines growth arrested at the G
1
-S-phase transition of the cell
cycle. Interestingly, a small percentage of the arrested Dodc
population remained viable 7–8 weeks after putrescine with-
drawal (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 Saccharo-
myces 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).
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 es-
tablished. Removal of Dodc 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 ODC
n/h
parasites in the absence of
exogenous polyamines also prompted a rapid decrease in the
FIG.7.Polyamine and thiol pools in ODC
n/h
cells incubated in
medium lacking polyamine. ODC
n/h
cells were removed from DME-L
supplemented with 200
m
M 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 3 10
8
cells were
removed for putrescine (●) and spermidine (E) measurements (A), and
10
8
cells were removed for the determination of trypanothione (f),
glutathionylspermidine (M), and glutathione (Œ) pools (B). Cell density
was monitored by Coulter counting (✕).
FIG.8.Polyamine catabolism in L. donovani. ODC
1/1
L. dono-
vani were incubated with [
14
C]spermidine (A)or[
14
C]spermine (B)as
reported under “Materials and Methods.” Cells were harvested and
washed, and polyamines were fractionated as described. 1.0-ml frac-
tions were then counted by liquid scintillation. The migration of known
standards of putrescine (PUT), spermidine (SPD), and spermine (SPN)
is indicated.
ODC Deficiency in L. donovani3786
cellular levels of trypanothione, which were subsequently
maintained at a low level, whereas concentrations of glutathi-
one 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 eleva-
tion in putrescine-depleted ODC
n/h
L. donovani parallels re-
sults 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.
Circumvention of the conditionally lethal Dodc 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 re-
duction 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 ac-
tivity. Moreover, exogenous spermine does not satisfy the poly-
amine requirement of the Dodc 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 N
1
-
acetyltransferase and polyamine oxidase. The inability of Dodc
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 N
1
-acetyl-
transferase/polyamine oxidase pathway altogether. This was
then confirmed radiometrically using both [
14
C]spermidine and
[
14
C]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 sper-
midine pools in L. donovani is governed exclusively by spermi-
dine synthase activity, unlike mammalian cells, in which in-
tracellular polyamines are regulated by a delicate balance
among the anabolic enzymes, spermidine and spermine syn-
thase, and the catabolic enzymes, spermidine/spermine N
1
-
acetyltransferase and polyamine oxidase. Although the poly-
amine 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 con-
junction with exogenous spermidine/spermine in therapies.
The polyamine requirement of Dodc cells can also be fulfilled
by either 1,3-diaminopropane or cadaverine, because the poly-
amine auxotrophs can grow continuously in DME-L-CS supple-
mented with either diamine, and each is taken up and accu-
mulated 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 essen-
tial metabolite for L. donovani. Small quantities of spermidine,
however, were observed in the 1,3-diaminopropane- and cadav-
erine-propagated cultures. The source of this spermidine is
unclear, although it may be present in the supplemented cul-
ture medium in insufficient amounts to support the growth of
the Dodc cells. Glutathionylspermidine and trypanothione lev-
els were also markedly reduced compared with either wild type
parasites or Dodc cells propagated in putrescine-supplemented
medium. Although previous investigators detected significant
amounts of homotrypanothione, the cadaverine-containing an-
alog 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 envi-
ronmental insult.
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 in-
duce protective immunity in mice and do not precipitate dis-
ease (42). We are currently evaluating the ability of our Dodc to
infect and proliferate within human macrophages, the cell type
in which the human stage of the parasite resides.
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