Amplification of adenine phosphoribosyltransferase suppresses the conditionally lethal growth and virulence phenotype of Leishmania donovani mutants lacking both hypoxanthine-guanine and xanthine phosphoribosyltransferases

Article abstract of DOI:10.1074/jbc.M110.125393

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084-16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice.

Full text of DOI:10.1074/jbc.M110.125393

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 18555–18564, June 11, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Amplification of Adenine Phosphoribosyltransferase
Suppresses the Conditionally Lethal Growth and Virulence
Phenotype of Leishmania donovani Mutants Lacking Both
Hypoxanthine-guanine and Xanthine
Phosphoribosyltransferases*
Received for publication, March 19, 2010, and in revised form, March 31, 2010 Published, JBC Papers in Press, April 2, 2010, DOI 10.1074/jbc.M110.125393
Jan M. Boitz and Buddy Ullman1
From the Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239
Leishmania donovani cannot synthesize purines de novo
and obligatorily scavenge purines from the host. Previously,
we described a conditional lethal ⌬hgprt/⌬xprt mutant of L.
donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem.
281, 16084–16089) that establishes that L. donovani salvages
purinesprimarilythroughhypoxanthine-guaninephosphoribosyl-
transferase (HGPRT) and xanthine phosphoribosyltransferase
(XPRT). Unlike wild type L. donovani, the ⌬hgprt/⌬xprt knock-
out cannot grow on 6-oxypurines and displays an absolute
requirement for adenine or adenosine and 2؅-deoxycoformycin,
an inhibitor of parasite adenine aminohydrolase activity. Here,
we demonstrate that the ability of ⌬hgprt/⌬xprt parasites to
infect mice was profoundly compromised. Surprisingly, mutant
parasites that survived the initial passage through mice partially
regained their virulence properties, exhibiting a >10-fold
increase in parasite burden in a subsequent mouse infection. To
dissect the mechanism by which ⌬hgprt/⌬xprt parasites per-
sisted in vivo, suppressor strains that had regained their capacity
to grow under restrictive conditions were cloned from cultured
⌬hgprt/⌬xprt parasites. The ability of these suppressor clones to
grow in and metabolize 6-oxypurines could be ascribed to a
marked amplification and overexpression of the adenine phos-
phoribosyltransferase (APRT) gene. Moreover, transfection of
⌬hgprt/⌬xprt cells with an APRT episome recapitulated the
suppressor phenotype in vitro and enabled growth on 6-oxypu-
rines. Biochemical studies further showed that hypoxanthine,
unexpectedly, was an inefficient substrate for APRT, evidence
that could account for the ability of the suppressors to metabo-
lize hypoxanthine. Subsequent analysis implied that APRT
amplification was also a potential contributory mechanism by
which ⌬hgprt/⌬xprt parasites displayed persistence and
increased virulence in mice.
within the alimentary tract and salivary glands of their insect
vector, members of the Phlebotomine sandfly family and as
nonflagellated, amotile amastigotes within macrophages and
other reticuloendothelial cells of the mammalian host. No
effective vaccines are available for visceral leishmaniasis—or
for that matter any disease caused by protozoan parasites, and
therefore chemotherapy offers the only means of defense for
the treatment and prevention of leishmaniasis and other dis-
eases of parasitic origin. Unfortunately, the current armamen-
tarium of drugs employed against visceral and other forms of
leishmaniasis is far from ideal and is adversely affected by
toxicity, protracted and invasive routes of administration,
and therapeutic unresponsiveness. As a result, there is an acute
need for better and more efficacious drugs to combat the
disease.
The establishment of an efficacious, parasite-specific reg-
imen for the treatment and prophylaxis of leishmaniasis
and other diseases of parasitic origin is contingent upon the
exploitation of fundamental biochemical or metabolic discrep-
ancies between the parasite and host. Perhaps the most striking
metabolic distinction between protozoan parasites and their
mammalian hosts are the pathways by which they produce
purine nucleotides. Whereas mammalian cells synthesize
purine nucleotides from amino acids and other small mole-
cules, protozoan parasites are incapable of synthesizing the
purine ring de novo (1–3). Thus, each genus of parasite has
evolved a unique complement of purine salvage enzymes that
enables the parasite to scavenge preformed purine bases and
nucleosides from its host. L. donovani accommodates four
enzymes that are capable of converting host purines to the
nucleotide level: hypoxanthine-guanine phosphoribosyltrans-
ferase (HGPRT),2 xanthine phosphoribosyltransferase (XPRT),
adenine phosphoribosyltransferase (APRT), and adenosine
kinase (2–4). Genetic studies of the purine pathway in L. dono-
vani have revealed that none of these four enzymes is, by itself,
essential for purine salvage, because mutant parasites deficient
in any one of the four enzymes are perfectly viable and exhibit
Leishmania donovani is a protozoan parasite that is the caus-
ative agent of visceral leishmaniasis, a debilitating and often
fatal disease in humans. Leishmania spp. are digenetic proto-
zoan parasites that exist as flagellated, motile promastigotes
2 The abbreviations used are: HGPRT, hypoxanthine-guanine phosphoribo-
syltransferase; XPRT, xanthine phosphoribosyltransferase; APRT, adenine
phosphoribosyltransferase; dCF, 2Ј-deoxycoformycin; DME-L, Dulbecco’s
modified Eagle’s Leishmania; FBS, fetal bovine serum; PBS, phosphate-
bufferedsaline;PFGE, pulsefieldgelelectrophoresis;Ni-NTA, nickel-nitrilo-
triacetic acid.
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant AI023682.
1 To whom correspondence should be addressed: 3181 S.W. Sam Jackson
Park Rd., Portland, OR 97239-3098. Fax: 503-494-8393; E-mail: ullmanb@
ohsu.edu.
JUNE 11, 2010•VOLUME 285•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 18555

Suppression of a Lethal Phenotype in Leishmania
no growth defects (5–9). The construction and characteriza- plete Mini EDTA-free protease inhibitor was procured from
tion of a conditionally lethal ⌬hgprt/⌬xprt null mutant using Roche Applied Science. Ni-NTA-agarose beads were from Qia-
targeted gene replacement approaches that exhibit patently gen, whereas the BiosafeTM Coomassie and protein assay kits
atypical growth requirements provided powerful genetic evi- were procured from Bio-Rad. All other chemicals and reagents
dence for the hypothesis that all salvage of purine nucleobases were of the highest quality commercially available.
and nucleosides by L. donovani ultimately occurs through
Parasite Cell Culture—The wild type LdBob L. donovani
HGPRT or XPRT and that APRT and adenosine kinase are clone (17) was obtained from Dr. Stephen Beverley (Washing-
functionally redundant (10). Whereas wild type L. donovani can ton University, St. Louis, MO). LdBob was derived from the
proliferate in virtually any purine nucleobase or nucleoside (2, 1S2D strain (18, 19) that had been acclimated for growth as
3, 11), the ⌬hgprt/⌬xprt mutant exhibits an absolute require- axenic amastigotes (17, 20). The construction and character-
ment for adenine or adenosine as a purine source and 2Ј-deoxy- ization of the ⌬hgprt/⌬xprt knock-out clone that was derived
coformycin (dCF), an inhibitor of the leishmanial adenine ami- from LdBob by targeted gene replacement and its episomally
nohydrolase enzyme (10, 12). Unlike wild type L. donovani, the complemented derivative ⌬hgprt/⌬xprt[pXPRT] have been
⌬hgprt/⌬xprt parasites cannot grow without 2Ј-deoxycoformy- reported previously (10). Wild type, ⌬hgprt/⌬xprt, and ⌬hgprt/
cin or with hypoxanthine, guanine, xanthine, guanosine, ⌬xprt[pXPRT] promastigotes were cultured at 26 °C, pH 7.4, in
inosine, or xanthosine as the sole purine nutrient (10). In addi- purine-replete modified Dulbecco’s modified Eagle’s Leishma-
tion, this double knock-out is, for all practical purposes, nonin- nia (DME-L) medium, as detailed (7), that was supplemented
fectious in mammalian macrophages (10). Both the condition- with 5% fetal bovine serum (FBS) or 5% dialyzed fetal bovine
ally lethal growth phenotype and the infectivity defect of the serum (7). Axenic amastigote forms of wild type, ⌬hgprt/⌬xprt,
⌬hgprt/⌬xprt knock-out can be circumvented genetically by and ⌬hgprt/⌬xprt[pXPRT] parasites were cultured at 37 °C, pH
episomal complementation with either HGPRT or XPRT or 5.5, in the synthetic medium as described (17, 20). The ⌬hgprt/
pharmacologically by maintenance in dCF plus adenine or ⌬xprt clone was routinely maintained as both promastigotes
adenosine as the exogenous purine (10).
and axenic amastigotes in 100 ␮M adenine as a purine and 20
We now report that the ability of the ⌬hgprt/⌬xprt double ␮M dCF, whereas the ⌬hgprt/⌬xprt[pXPRT] “add-back” strain
null mutant to infect Balb/c mice, a well characterized rodent was cultured in 100 ␮M xanthine without dCF and 50 ␮g/ml
model for visceral leishmaniasis (13–16), is profoundly com- blasticidin to maintain selective pressure for episome expres-
promised. This virulence deficit, however, is partially amelio- sion. Single cell cloning protocols for L. donovani promastig-
rated in ⌬hgprt/⌬xprt parasites that persist through a 4-week otes have been described (21).
infection in mice. To investigate this persistent phenotype fur-
Mouse Infections—Groups of five 7-week-old female Balb/c
ther, we isolated second site suppressors of the ⌬hgprt/⌬xprt mice (Charles River Laboratories, Wilmington, MA) were inoc-
mutant under controlled circumstances by exposing the knock- ulated by tail vein injection with 5 ϫ 106 of either wild type,
out parasites to a variety of nonpermissive growth conditions ⌬hgprt/⌬xprt, or ⌬hgprt/⌬xprt[pXPRT] stationary phase pro-
in vitro. ⌬hgprt/⌬xprt parasites (⌬hgprt/⌬xprt[Ino/Hyp]) that mastigotes (10, 16). Prior to injection, each L. donovani strain
could be maintained in inosine, hypoxanthine, adenine, or was cycled back and forth several times between promastigote
adenosine in the absence of dCF were isolated after two rounds and axenic amastigote forms (20) to revitalize ancillary viru-
of selection. We determined that the suppressor mechanism by lence determinants that might have attenuated as a result of
which these ⌬hgprt/⌬xprt[Ino/Hyp] parasites could survive prolonged in vitro culture. Four weeks post-infection, the mice
under conditions that were restrictive for the ⌬hgprt/⌬xprt were sacrificed, and their livers and spleens were harvested as
progenitor was amplification and overexpression of the APRT reported (16). Single-cell suspensions from the mouse organs
gene. Moreover, the ⌬hgprt/⌬xprt[Ino/Hyp] growth phenotype were obtained by passage through a 70-␮m cell strainer (BD
could be reconstructed by transfection of an episomal APRT Falcon, Franklin Lakes, NJ), and the parasite burdens were
construct into the conditional lethal ⌬hgprt/⌬xprt mutant. determined in 96-well microtiter plates employing the limiting
Further analysis of the ⌬hgprt/⌬xprt parasites that persisted dilution assay of Buffet et al. (22). The growth medium in which
through the mouse infection implied that APRT amplification the organ-derived wild type, ⌬hgprt/⌬xprt, and ⌬hgprt/⌬xprt-
was also likely operative in parasite persistence in vivo.
[pXPRT] parasites were titered was modified DME-L (7) sup-
plemented with 5% FBS and 100 ␮M adenine, 100 ␮M adenine
plus 20 ␮M dCF, or 100 ␮M xanthine, respectively. Four weeks
EXPERIMENTAL PROCEDURES
Materials, Chemicals, and Reagents—[8-14C]Adenine (50 after harvest, wild type and persistent ⌬hgprt/⌬xprt parasites
mCi/mmol) and [8-14C]hypoxanthine (51 mCi/mmol) were that survived the initial infection were reinoculated into a naïve
purchased from Moravek Biochemicals (Brea, CA). dCF was group of mice. Fifteen mice were infected with each strain, and
obtained from the National Cancer Institute (Bethesda, MD). three mice from each group were sacrificed at 2-week time
Unlabeled purine bases, nucleosides, and nucleotides, were intervals beginning at week 2 and ending at week 10. Parasites
bought from Sigma-Aldrich and Fisher. Mouse monoclonal recovered from livers and spleens after the second round of
anti-␣-tubulin antibody was obtained from Calbiochem/EMD infection were enumerated as described above, and the 4-week
Biosciences Inc. (La Jolla, CA), and anion exchange filters time points from each mouse experiment were compared.
were acquired from Whatman. The ChampionTM pET200/D-
Selection for Suppressor Mutants in Vitro—Parasite lines that
TOPO௡ expression vector and BL21 StarTM (DE3) One Shot௡ had suppressed the restricted growth phenotype of the ⌬hgprt/
competent cells were purchased from Invitrogen, and the Com- ⌬xprt null mutant were isolated by plating knock-out cells
18556 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 24•JUNE 11, 2010

Suppression of a Lethal Phenotype in Leishmania
under nonpermissive growth conditions, i.e. in a 6-oxypurine
source in the absence of dCF, as follows. 5 ϫ 107 ⌬hgprt/⌬xprt
promastigotes were plated on semi-solid DME-L medium
containing either adenine, adenosine, hypoxanthine, ino-
sine, guanine, guanosine, xanthine, or xanthosine, all at 100
␮M concentrations, and supplemented with 20% dialyzed
FBS. dCF was omitted in these selective platings. Four clones,
designated ⌬hgprt/⌬xprt[Ino], were picked from the inosine
plates and expanded in modified DME-L containing 100 ␮M
inosine and 5% FBS. Two of the ⌬hgprt/⌬xprt[Ino] clones were
then replated on semi-solid modified DME-L medium supple-
mented with 20% dialyzed FBS and 100 ␮M purine in the
absence of dCF. After this second round of selection, six clones
were isolated from the plates containing 100 ␮M hypoxanthine
as the exclusive purine. These cells were designated ⌬hgprt/
⌬xprt[Ino/Hyp].
Immunoblotting and DNA Manipulations—Monospecific
polyclonal antibodies raised against purified recombinant L.
donovani APRT, HGPRT, and XPRT proteins in rabbits have
been described previously (23–25), and Western blotting
protocols were performed as detailed (26). Monoclonal anti-
␣-tubulin antibody (DM1A) produced in mice was obtained
from EMD Chemicals (Gibbstown, NJ). Goat anti-rabbit
and goat anti-mouse secondary antibodies were purchased
from Thermo Fisher Scientific Pierce Protein Research Prod-
ucts (Rockford, IL). Isolation of genomic DNA and Southern
blot analysis were accomplished using conventional protocols
(26). The previously utilized (7, 10) hybridization probes har-
boring the full-length L. donovani APRT, HGPRT, and XPRT
open reading frames (7, 10, 27) were amplified by polymerase
chain reaction from a TOPO-TA PCR 2.1௡ vector (Invitrogen)
containing the full-length APRT, HGPRT, or XPRT coding
sequence and gel-purified using a Wizard SV gel and PCR
clean-up kit (Promega, Madison, WI).
Growth Phenotypes—To assess the abilities of the wild type,
⌬hgprt/⌬xprt, ⌬hgprt/⌬xprt[Ino], and ⌬hgprt/⌬xprt[Ino/Hyp]
promastigotes to grow in different purine sources, exponen-
tially growing parasites were washed several times with phos-
phate-buffered saline (PBS), resuspended at a density of 5 ϫ 104
cells/ml in 1.0-ml aliquots of modified DME-L (7) containing
100 ␮M purine and 5% dialyzed FBS, and dispensed into wells of
24-well tissue culture plates (Sarstedt Inc., Newton, NC). After
7–10 days, the parasites were enumerated by hemocytometer.
Macrophage Infections—Peritoneal macrophages from
Balb/c mice were harvested 5 days after induction by thiogly-
collate injection (16), washed twice in PBS, and resuspended in
Dulbecco’s modified Eagle’s medium supplemented with 4 mM
L-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose,
and 10% FBS. 2 ϫ 105 macrophages/well were allowed to
adhere for ϳ12 h at 37 °C to 4-well Lab-TekII chamber slides
and then washed once with PBS and replenished with fresh
growth medium. Stationary phase wild type, ⌬hgprt/⌬xprt,
and ⌬hgprt/⌬xprt[Ino/Hyp] promastigotes were washed
twice in PBS and resuspended in macrophage medium, and 2 ϫ
106 parasites were added to each chamber slide well of adherent
macrophages and incubated at 37 °C. Residual extracellular
promastigotes were removed by gently washing the macro-
phages three times with PBS 12 h post-infection. The macro-
phages were rinsed with PBS, and their growth medium was
changed daily. After 72 h the macrophages were washed,
stained, and enumerated as described (7, 16).
Immunofluorescence Assay—ϳ5 ϫ 106 L. donovani promas-
tigotes were pelleted by centrifugation, washed once with PBS,
resuspended in 1 ml of PBS, and ultimately affixed to four-well
Lab-Tek௡II chamber slides (Nalge Nunc International, Roches-
ter, NY) that had been treated with 10% poly-L-lysine. The
immunofluorescence assay was carried out as described (27, 28)
using a 1:100 dilution of recombinant anti-APRT antibody and
a 1:1000 dilution of secondary goat anti-rabbit antibody conju-
gated to Oregon Green (Invitrogen) that was applied in a block-
ing buffer containing 3% goat serum. The cells were visualized
on a Zeiss Axiovert 200 inverted microscope (Carl Zeiss Micro-
imaging, Thornwood, NY) employing 60ϫ oil immersion light
and photographed with an AxioCam MRm camera (Zeiss).
Axiovision 4.2 software was used to photograph the images.
Expression of APRT in Escherichia coli—The APRT open
reading frame was amplified by PCR from the previously
described [pXG-BSD-APRT] episome (7) and inserted into the
bacterial expression vector ChampionTM pET200/D-TOPO௡
that automatically attaches a His6 tag to the NH2 terminus of
the inserted gene product. The forward primer that was used to
amplify APRT included the sequence CACC before the APRT
ATG start codon to allow directional cloning into the pET200/
D-TOPO௡ vector. The amplified DNA construct was then
sequenced bidirectionally to ensure the fidelity of the PCR
amplification. The chimeric construct was transformed into
BL21 StarTM One Shot௡ E. coli, and the bacterial culture was
induced with 1.0 mM isopropyl-␤-D-thiogalactopyranoside to
synthesize APRT protein from the plasmid as described (29).
Purification of Recombinant His6-APRT—His6-tagged L.
donovani APRT was purified by affinity chromatography over a
Ni-NTA-agarose (Qiagen) column from E. coli extracts that
were prepared by means of a French press as described (29)
except that the concentration of imidazole in the final wash
buffer was increased from 20 to 30 mM. Recombinant LdAPRT
was eluted from the Ni-NTA-agarose with 250 mM imidazole as
detailed (29). Separation of the purified recombinant APRT
fractions on a 10% SDS-polyacrylamide gel and subsequent
staining with Bio-safeTM Coomassie (Bio-Rad) confirmed the
purity of the recombinant protein. A Thermo Labsystems Mul-
tiskan Ascent plate reader was employed at 600 nm to deter-
Hypoxanthine Incorporation by Intact Parasites—The ability
of intact parasites to convert [8-14C]hypoxanthine into purine
nucleotides was determined by the DE-81 filter disk method of
Iovannisci et al. (8). Briefly, L. donovani promastigotes were
harvested by centrifugation, washed twice in PBS, and resus-
pended at a density of 1.0 ϫ 108 cells/ml in 1.0 ml of a modified
DME-L medium containing 2 ␮M [8-14C]hypoxanthine (51
mCi/mmol) but lacking albumin, FBS, and hemin. At each time
point, 1.0 ϫ 107 parasites were removed, washed once in ice
cold PBS, lysed in 50 ␮l of 1% Triton X-100, and spotted onto a
DE-81 filter disk (8). The disks were processed as described
(6–8, 10) and air-dried, and incorporation of [8-14C]hypoxan-
thine into phosphorylated metabolites was quantified by liquid
scintillation spectrometry.
JUNE 11, 2010•VOLUME 285•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 18557

Suppression of a Lethal Phenotype in Leishmania
mine the protein concentration and yield of the purified APRT
after the addition of Bio-Rad Protein Assay reagent (29). The
catalytic activity of purified recombinant APRT protein was
ascertained immediately following purification.
APRT Assays—To determine the linear rate of conversion of
hypoxanthine to IMP, 10 ␮g of purified, recombinant LdAPRT
was added to reaction buffer containing 20 mM Tris-HCl, pH
7.4, 5 mM MgCl2, 10 mM NaF, 1 mM phosphoribosylpyrophos-
phate, and 57 ␮M [8-14C]hypoxanthine (51 mCi/mmol). The
final reaction volume was 35 ␮l. At each time point over a 2-h
time course, 5-␮l aliquots were mixed with 2 ␮l of glacial acetic
acid to terminate the reaction (30), spotted onto a Whatman PE
SIL G silica gel TLC plate, and developed in dioxane/ammo-
nium hydroxide/water 6:1:5 (v/v/v) (31). The amount of IMP
produced was quantified using a Bioscan AR-2000 plate reader
and Bioscan Winscan two-dimensional software (Bioscan Inc.,
Washington DC). Similarly, when adenine was the substrate,
0.1 ␮g of purified, recombinant LdAPRT was added to reaction
buffer containing 57 ␮M [8-14C]adenine (50 mCi/mmol). After
mixing with glacial acetic acid, the reaction mix was spotted
onto a DE-81 filter disk at each time point over a 5-min time
course, and the disks were counted on a scintillation counter to
quantify the amount of AMP produced.
FIGURE 1. Parasite burdens in livers and spleens of mice infected with
wild type, ⌬hgprt/⌬xprt, and add-back parasites. A, three separate groups
of five Balb/c mice were infected with either wild type, ⌬hgprt/⌬xprt, or
⌬hgprt/⌬xprt[pXG-BSD-XPRT] (⌬hgprt/⌬xprt[pXPRT]) stationary phase pro-
mastigotes. The mice were sacrificed 4 weeks post-infection, and the parasite
loads in livers and spleens were quantified using limiting dilution. The limit-
ing dilution medium for wild type and (⌬hgprt/⌬xprt[pXPRT] parasites con-
tained 100 ␮M xanthine as the purine source, whereas ⌬hgprt/⌬xprt were
quantified by growth under permissive conditions consisting of 100 ␮M ade-
nine and 10 ␮M dCF. B, two groups of five naïve mice were infected with wild
type or ⌬hgprt/⌬xprt parasites harvested after the initial infection from
mouse livers. Limiting dilution was employed 4 weeks post-inoculation to
verify the parasite load. The medium was supplemented with the same
purines as in A.
Michaelis-Menten kinetics were determined using the lin-
ear rate of conversion of hypoxanthine to IMP over a 1-h
time course. Either 28.5 ␮M [8-14C]hypoxanthine or 57 ␮M
[8-14C]hypoxanthine was mixed with nonradiolabeled hypox-
anthine to a final concentration between 50 ␮M and 5 mM. TLC
was used to separate the radiolabeled products, and the amount
of IMP produced was quantified as described above.
Pulse Field Gel Electrophoresis (PFGE)—InCert-agarose
(Cambrex, Rockland, ME) plugs (5%) containing 2 ϫ 107 L.
donovani promastigotes were prepared as described (32, 33).
Chromosomes of wild type, ⌬aprt, ⌬hgprt/⌬xprt, and two inde-
pendent clones of ⌬hgprt/⌬xprt[Ino/Hyp] parasites were frac- weeks post-infection, and the parasite load within the infected
tionated by PFGE using a contour-clamped homogeneous elec- livers and spleens was determined by limiting dilution. The para-
tric field gel apparatus (Bio-Rad) on a 1% agarose gel at 14 °C for site burdens (parasites/g of tissue) in the livers and spleens of mice
24 h with a 60-s pulse time in 0.5ϫ Tris-borate-EDTA buffer as infected with wild type L. donovani were ϳ10,000- and ϳ100-fold
described (33). The gel was stained with ethidium bromide, and higher, respectively, than those from mice infected with the
a conventional Southern blot was performed (26) using 32P- ⌬hgprt/⌬xprt knock-out (Fig. 1A). The virulence defect was
labeled full-length APRT coding sequence as the probe (7).
rescued almost completely by complementation with an XPRT
Construction of Transgenic ⌬hgprt/⌬xprt Parasites Comple- episome, because the parasite loads in mice infected with
mented with LdAPRT—The pXG-BSD-APRT episome (7) was ⌬hgprt/⌬xprt[pXPRT] add-back parasites were virtually indis-
transfected into ⌬hgprt/⌬xprt cells as described (17), and the tinguishable from those infected with wild type parasites.
parasites were plated on semi-solid growth medium containing
Although the virulence of ⌬hgprt/⌬xprt parasites in mice
20% dialyzed FBS and 100 ␮M hypoxanthine. Several ⌬hgprt/ was severely compromised, a small number of null mutant
⌬xprt[pAPRT] colonies were picked and expanded in modified parasites persisted through the duration of the mouse infec-
DME-L supplemented with 5% dialyzed FBS and 100 ␮M tions. To evaluate whether the persistent population pos-
hypoxanthine.
sessed extraordinary virulence properties, a second round of
infection in naïve mice was performed with wild type and
⌬hgprt/⌬xprt parasites isolated from the first cycle of infection.
RESULTS
Virulence Defect of ⌬hgprt/⌬xprt Parasites in Mice—Because Parasite burdens in the livers and spleens of mice infected with
⌬hgprt/⌬xprt L. donovani are effectively noninfectious in survivors from the first wild type infection were effectively
murine peritoneal macrophages (10), the ability of the double equivalent to those achieved in the first series of infections (Fig.
knock-out to infect Balb/c mice, a well characterized rodent 1). However, the persistent ⌬hgprt/⌬xprt parasites from the
model for leishmaniasis (13–16), was evaluated. The mice first infection cycle exhibited markedly elevated parasite loads
were inoculated with wild type, ⌬hgprt/⌬xprt, or ⌬hgprt/ as compared with the ⌬hgprt/⌬xprt parasites in the first
⌬xprt[pXPRT] parasites via tail vein injection and sacrificed 4 sequence of mouse infections (Fig. 1). The increase in ⌬hgprt/
18558 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 24•JUNE 11, 2010

Suppression of a Lethal Phenotype in Leishmania
survive after 4 weeks in a mamma-
lian host, we attempted to recreate
the persistent phenotype observed
in the null parasites passaged
through mice by isolating suppres-
sor parasites in vitro under restric-
tive growth circumstances. The
⌬hgprt/⌬xprt knock-out, which is
only capable of sustained and rapid
growth in adenine/adenosine in the
presence of dCF, was subjected to
two rounds of selection under non-
permissive conditions (Fig. 2). No
⌬hgprt/⌬xprt parasites survived
the first round of selection on
plates containing adenine, adeno-
sine, hypoxanthine, guanine, gua-
nosine, xanthine, or xanthosine as
the sole purine. No dCF was added
to any of these plates. In contrast,
four viable ⌬hgprt/⌬xprt[Ino] colo-
nies were obtained from plates
containing inosine as a purine
source, and one of these clones
was subjected to further analysis.
FIGURE2. Growthphenotypesof⌬hgprt/⌬xprt[Ino]and⌬hgprt/⌬xprt[Ino/Hyp]suppressors. A,wildtype,
Two ⌬hgprt/⌬xprt[Ino] clones
were expanded further in DME-L
supplemented with inosine and
replated in semi-solid DME-L con-
taining the same purines as speci-
fied above for the selections of the
⌬hgprt/⌬xprt, and ⌬hgprt/⌬xprt[Ino] promastigotes were incubated in growth medium containing the indi-
cated purine additions or no purine, and the parasite numbers were quantified by hemocytometer. B, wild
type, ⌬hgprt/⌬xprt, and ⌬hgprt/⌬xprt[Ino/Hyp]promastigoteswerecomparedfortheircapabilitiesofgrowing
in the same purines specified in A. The results depicted in the figure are the averages and standard errors of
three replicates. Ade, adenine; Ado, adenosine; Gua, guanine; Guo, guanosine; Hyp, hypoxanthine; Ino, inosine;
Xan, xanthine; Xao, xanthosine.
original ⌬hgprt/⌬xprt[Ino] clones. In this second round of plat-
ing, several colonies of ⌬hgprt/⌬xprt[Ino] parasites were
obtained on plates containing inosine, adenine, adenosine, or
hypoxanthine. Onceagain, dCFwasomittedfromtheseselections.
The six clones picked from the hypoxanthine-containing plates
were designated ⌬hgprt/⌬xprt[Ino/Hyp], and two of them,
clones 3-1 and 4-1, were chosen for further analysis. Interest-
ingly, no ⌬hgprt/⌬xprt[Ino/Hyp] progeny were obtained from
plates containing guanine, guanosine, xanthine, or xanthosine.
Growth Phenotypes of ⌬hgprt/⌬xprt[Ino] and ⌬hgprt/⌬xprt[Ino/
Hyp] Parasites—The capacities of wild type ⌬hgprt/⌬xprt,
⌬hgprt/⌬xprt[Ino] (Fig. 2A), and ⌬hgprt/⌬xprt[Ino/Hyp] (Fig.
2B) promastigotes to proliferate in various purine sources
were compared. Whereas wild type L. donovani could utilize
adenine, adenosine guanine, guanosine, hypoxanthine,
inosine, xanthine, or xanthosine as its purine nutrient, the
⌬hgprt/⌬xprt knock-out only grew in adenine or adenosine in
the presence of 20 ␮M dCF (Fig. 2). Both the ⌬hgprt/⌬xprt[Ino]
FIGURE 3. Parasitemia of wild type, null, and ⌬hgprt/⌬xprt[Ino/Hyp] sup-
pressor parasites in peritoneal murine macrophages. Mouse peritoneal
macrophages were infected with wild type, ⌬hgprt/⌬xprt, or ⌬hgprt/
⌬xprt[Ino/Hyp] clone 4-1 stationary phase promastigotes at a ratio of 10 par-
asites/macrophage. The cells were stained after 72 h, and the amastigotes
were enumerated visually. The results are the averages and standard errors of
four independent determinations (n ϭ 4).
⌬xprt parasite loads in the second set of infections compared and ⌬hgprt/⌬xprt[Ino/Hyp] lines, however, exhibited less
with the first was approximately 1 and 2 orders of magnitude for restricted growth phenotypes than the ⌬hgprt/⌬xprt null
livers and spleens, respectively. Parasite numbers of the persis- mutant from which they were derived. The ⌬hgprt/⌬xprt[Ino]
tent ⌬hgprt/⌬xprt parasites in the second round of infection and ⌬hgprt/⌬xprt[Ino/Hyp] cells lines were now capable of sus-
were only 750-fold less than those of wild type parasites in liver, tained proliferation in inosine and could grow in adenine or
whereas splenic parasitemias between wild type and knock-out adenosine without dCF supplementation (Fig. 2). In addition,
parasites were comparable (Fig. 1B).
the ⌬hgprt/⌬xprt[Ino/Hyp] but not the ⌬hgprt/⌬xprt[Ino] cells
Isolation of ⌬hgprt/⌬xprt[Ino/Hyp] Suppressors—To dissect could utilize hypoxanthine as the purine nutrient (Fig. 2). It is
the mechanism by which persistent ⌬hgprt/⌬xprt parasites important to note, however, that the growth exhibited by the
JUNE 11, 2010•VOLUME 285•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 18559

Suppression of a Lethal Phenotype in Leishmania
plus dCF, was equivalent. As an
example of the slow growth pheno-
type, the doubling time of the ⌬hgprt/
⌬xprt[Ino/Hyp] line in hypoxanthine
was ϳ16 h compared with ϳ10 h for
wild type parasites grown under the
same conditions.
⌬hgprt/⌬xprt[Ino/Hyp] L. dono-
vani Can Infect Macrophages—Be-
cause the ⌬hgprt/⌬xprt null mutant
is profoundly incapacitated in its
ability to achieve a robust infection
in macrophages (10) and mice (Fig.
1), the ability of a ⌬hgprt/⌬xprt[Ino/
Hyp] suppressor to infect macro-
phages was evaluated (Fig. 3). The
parasite burden of the ⌬hgprt/
⌬xprt[Ino/Hyp] line in peritoneal
murine macrophages was ϳ13
amastigotes/macrophage, a parasite
load comparable with that of wild
type parasites. In contrast, only ϳ1
amastigote/macrophage was recov-
ered for the ⌬hgprt/⌬xprt null
mutant (Fig. 3). The ability of the
⌬hgprt/⌬xprt[Ino] line to infect
macrophages was not tested.
FIGURE 4. Purine incorporation into intact L. donovani promastigotes. The abilities of intact wild type (f),
⌬hgprt/⌬xprt (F), and two independent ⌬hgprt/⌬xprt[Ino/Hyp] (Œ and ꢀ) lines to incorporate 28 ␮M [14C]hy-
poxanthine into nucleotides were measured over a 3-h time course as described under “Experimental
Procedures.”
⌬hgprt/⌬xprt[Ino/Hyp] Cells Me-
tabolize Hypoxanthine—Because
⌬hgprt/⌬xprt[Ino/Hyp] cells reac-
quired the capability of growing,
albeit slowly, with hypoxanthine
as the sole exogenous purine, the
ability of intact wild type, ⌬hgprt/
⌬xprt, and ⌬hgprt/⌬xprt[Ino/Hyp]
parasites to incorporate [8-14C]hy-
poxanthine was assessed (Fig. 4).
Whereas wild type parasites dem-
onstrated robust incorporation of
the extracellular nucleobase into
intracellular nucleotides, as expected,
no measurable hypoxanthine metab-
olism was observed in ⌬hgprt/⌬xprt
cells. The two independent ⌬hgprt/
⌬xprt[Ino/Hyp] clones, however,
had regained the capacity to incor-
porate exogenous hypoxanthine
into the parasite nucleotide pool,
but not to wild type levels.
FIGURE 5. Western and Southern blot analysis of ⌬hgprt/⌬xprt[Ino/Hyp] parasites. 5 ϫ 106 parasites were
processed and loaded into each lane of an SDS-PAGE gel for Western analysis. A, lysates from 5 ϫ 106 wild type,
⌬hgprt/⌬xprt, ⌬aprt, ⌬hgprt/⌬xprt[Ino], and two ⌬hgprt/⌬xprt[Ino/Hyp] strains were fractionated by SDS-
PAGE and blotted with either anti-APRT, anti-HGPRT, or anti-XPRT polyclonal antisera. The amount of lysate
loadedintoeachlaneofthegelwasnormalizedwithmonoclonalmouseanti-␣-tubulinantisera. B, lysatesfrom
wild type, ⌬hgprt/⌬xprt, ⌬aprt, and ⌬hgprt/⌬xprt[Ino/Hyp]clone 4-1 (lanes 1–4) L. donovani and serial dilutions
of ⌬hgprt/⌬xprt[Ino/Hyp] clone 4-1 lysates (lanes 5–10) were subjected to Western blot analysis with anti-APRT
antibodyandnormalizedwithanti-␣-tubulinantisera. C, theAPRTgenecopynumberin⌬hgprt/⌬xprt[Ino/Hyp]
clone 4-1 parasites was evaluated by hybridizing genomic DNA prepared from wild type, ⌬hgprt/⌬xprt, ⌬aprt,
⌬hgprt/⌬xprt[Ino/Hyp]clone3-1, ⌬hgprt/⌬xprt[Ino/Hyp]clone4-1parasites(lanes1–5), aswellastheindicated
2-fold serial dilutions of ⌬hgprt/⌬xprt[Ino/Hyp] clone 4-1 (lanes 7–13) that had been digested with BamHI/SalI,
fractionated on an 0.8% agarose gel, blotted onto a nylon membrane, and probed with the full-length APRT
open reading frame. D, the ethidium bromide-stained gel of lanes 1–5 shows the relative amounts of DNA
loaded in C.
APRT Protein Overexpression in
⌬hgprt/⌬xprt[Ino] and ⌬hgprt/
⌬xprt[Ino/Hyp]—The ability of the
⌬hgprt/⌬xprt[Ino/Hyp] cells to
⌬hgprt/⌬xprt[Ino] and ⌬hgprt/⌬xprt[Ino/Hyp] lines in inosine, incorporate, salvage, and replicate in hypoxanthine was initially
adenine, or adenosine was also considerably more sluggish than a cause of concern because it could have theoretically been
wild type promastigotes grown in the same purines, whereas ascribed to contamination by wild type parasites. To allevi-
growth of wild type, ⌬hgprt/⌬xprt[Ino], and ⌬hgprt/⌬xprt[Ino/ ate this apprehension that the recovered metabolic capacity
Hyp] lines under completely permissive conditions, i.e. adenine of the ⌬hgprt/⌬xprt[Ino/Hyp] suppressor lines to take up
18560 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 24•JUNE 11, 2010

Suppression of a Lethal Phenotype in Leishmania
FIGURE 7. Western blot analysis of persistent ⌬hgprt/⌬xprt parasites har-
vested from mice. Extracts from wild type (lanes 1 and 2) and ⌬hgprt/⌬xprt
(lanes 3 and 4) parasite cultures that were harvested from mouse livers and
expanded under permissive growth conditions were subjected to Western
blot analysis using anti-APRT monospecific polyclonal antisera. The amounts
of cell lysate loaded onto each lane were normalized with ␣-tubulin antisera.
FIGURE 6. Immunofluorescence analysis of ⌬hgprt/⌬xprt[Ino/Hyp] para-
sites. Wild type (A) and ⌬hgprt/⌬xprt[Ino/Hyp] (D) suppressor promastigotes
were incubated with rabbit anti-APRT antisera and visualized at 488 nm using
goat anti-rabbit IgG Oregon Green-conjugated secondary antibody. B and E
depict wild type and ⌬hgprt/⌬xprt[Ino/Hyp] parasites, respectively, that have
been stained with 4Ј,6-diamidino-2-phenylindole for DNA visualization.
Phase contrast images of the stained parasites shown in A, B, D, and E are
shown in C and F.
also evaluated for elevated APRT protein levels. It should be
noted, however, that these persistent ⌬hgprt/⌬xprt parasites
were expanded and grown under permissive growth condi-
tions, i.e. adenine plus dCF, after rescue from mice. Western
blot analysis revealed that those persistent parasites that were
grown to high density in vitro in medium that did not select for
APRT amplification expressed slightly elevated amounts of
hypoxanthine may have been artifactually triggered through APRT protein, ϳ3-fold greater than the APRT level of wild type
incidental contamination by wild type cells, Western blot parasites (Fig. 7). The immunoblot was probed with antibody to
analysis was carried out with anti-HGPRT and anti-XPRT ␣-tubulin as the loading control.
antibodies on wild type, ⌬hgprt/⌬xprt, ⌬hgprt/⌬xprt[Ino],
APRT Utilizes Hypoxanthine as a Substrate—The L. dono-
and ⌬hgprt/⌬xprt[Ino/Hyp] cell extracts. These experi- vani APRT has been previously reported to exclusively recog-
ments demonstrated that ⌬hgprt/⌬xprt, ⌬hgprt/⌬xprt[Ino], nize adenine as a substrate (23, 34). However, the correlation
and ⌬hgprt/⌬xprt[Ino/Hyp] did not express detectable between the capacity of ⌬hgprt/⌬xprt[Ino/Hyp] to incorporate
HGPRT or XPRT protein (Fig. 5A). For the purpose of nor- and grow in hypoxanthine (Figs. 2 and 4) with the amplification
malizing the amount of parasite extract loaded onto each and overexpression of APRT (Figs. 5 and 6) implied that APRT
lane, the same immunoblots were also probed with antibod- was capable of phosphoribosylating hypoxanthine. To test this
ies against ␣-tubulin and APRT (23) (Fig. 5A). Surprisingly, conjecture, recombinant APRT was produced and purified. Ini-
the amount of APRT protein in ⌬hgprt/⌬xprt[Ino] and tial experiments demonstrated that APRT could recognize
⌬hgprt/⌬xprt[Ino/Hyp] promastigotes was strikingly higher hypoxanthine as a substrate but far less efficiently than adenine.
than that of wild type or ⌬hgprt/⌬xprt cells (Fig. 5A). APRT Indeed, detection of hypoxanthine phosphoribosylation by
protein from both ⌬hgprt/⌬xprt[Ino/Hyp] isolates was APRT required prolonged assay times and a high concentration
ascertained to be ϳ30-fold greater than in wild type parasites of recombinant protein. APRT-catalyzed hypoxanthine phos-
by densitometry. A quantitative Western blot is shown for phoribosylation was linear with time for up to 2 h (Fig. 8A). A
clone 4-1 (Fig. 5B). Immunofluorescence analysis of APRT 5-min time course for adenine is also shown for comparison
confirmed the conspicuous augmentation of APRT protein (Fig. 8B). The rates of conversion of hypoxanthine to IMP and
in the ⌬hgprt/⌬xprt[Ino/Hyp] parasites (Fig. 6). A cytosolic adenine to AMP were 0.1726 nmol/min/mg protein and 3.017
milieu for APRT in L. donovani promastigotes has been pre- ␮mol/min/mg protein, respectively, for the time courses shown
viously demonstrated (27).
(Fig. 8, A and B). APRT was saturable only at millimolar con-
APRT Gene Amplification in ⌬hgprt/⌬xprt[Ino] and ⌬hgprt/ centrations of the 6-oxypurine substrate hypoxanthine (Fig.
⌬xprt[Ino/Hyp] Cells—To examine the genetic changes 8C). Michaelis-Menten analysis revealed the apparent Km of
that transpired within the ⌬hgprt/⌬xprt[Ino] and ⌬hgprt/ APRT for hypoxanthine to be ϳ1.5 mM with a Vmax of ϳ1.3
⌬xprt[Ino/Hyp] genomes, Southern blotting was performed to nmol/min/mg protein (Fig. 8C) as compared with 1.2 ␮M and
establish whether the APRT gene had been amplified. This 17.5 ␮mol/min/mg protein, respectively, for adenine, as previ-
analysis revealed that APRT was greatly amplified in all of the ously demonstrated by Allen et al. (23). The catalytic efficien-
⌬hgprt/⌬xprt[Ino/Hyp] clones examined (data not shown). cies of APRT for adenine and hypoxanthine were calculated to
Quantification indicated ϳ30–50-fold amplification of APRT be 1.09 ␮MϪ1 sϪ1 and 3.51 ϫ 10 ␮MϪ1 sϪ1, respectively.
Ϫ7
in both ⌬hgprt/⌬xprt[Ino/Hyp] clones. The Southern blot for
⌬hgprt/⌬xprt[Ino/Hyp] Parasites Harbor Extrachromosomal
clone 4-1 is shown in Fig. 5C, and the DNA gel is shown to Elements Containing APRT—PFGE analysis of two ⌬hgprt/
indicate loading control (Fig. 5D). Genomic DNA from ⌬aprt ⌬xprt[Ino/Hyp] clones revealed that each possessed extrachro-
(7) parasites was included in this Southern analysis as a negative mosomal elements that were not present in wild type, ⌬aprt, or
control (Fig. 5, C and D).
⌬hgprt/⌬xprt parasites (Fig. 9). The most prominent of these
APRT Protein Levels Are Increased in ⌬hgprt/⌬xprt Parasites extrachromosomal elements in the two ⌬hgprt/⌬xprt[Ino/
That Persist in Mice—Because APRT amplification and overex- Hyp] clones could easily be envisioned by ethidium bromide
pression was observed in ⌬hgprt/⌬xprt[Ino/Hyp] parasites that staining and revealed molecular masses of ϳ200 and ϳ275
were obtained by selection in vitro, the persistent ⌬hgprt/⌬xprt kb, respectively (Fig. 9A). Hybridization of the pulse field gel
parasites that survived two rounds of infection in mice were to APRT revealed that the vast majority of the amplified
JUNE 11, 2010•VOLUME 285•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 18561

Suppression of a Lethal Phenotype in Leishmania
FIGURE 8. Reaction kinetics of APRT with hypoxanthine. A, 10 ␮g of purified, recombinant L. donovani APRT protein was incubated with [14C]hypoxanthine
over a 2-h time course, and the amount of IMP produced was measured as described under “Experimental Procedures.” B, 0.1 ␮g of enzyme was used to
determine the rate of conversion of [14C]adenine to AMP over a 5-min time course by APRT. C, recombinant L. donovani APRT protein was purified and
incubatedwithvariousconcentrationsof[14C]hypoxanthine, andthephosphorylatedproductwasquantitatedasdescribedunder“ExperimentalProcedures.”
The data points are the averages and standard deviations of three independent experiments. The kinetic constants were calculated using the Lineweaver-Burk
algorithm from GraphPad Prism.
[pXG-BSD-APRT] recreated a growth profile comparable with
that of the ⌬hgprt/⌬xprt[Ino/Hyp] suppressor strains (Fig. 10).
The ⌬hgprt/⌬xprt[pAPRT] transfectant, similar to the hgprt/
⌬xprt[Ino/Hyp] cells (Fig. 2), was capable of continuous growth
in adenine or adenosine in the absence of dCF, inosine, or
hypoxanthine, although the doubling time for ⌬hgprt/⌬xprt-
[pAPRT] parasites, as anticipated, was considerably slower than
when the cells were grown in the permissive conditions of ade-
nine or adenosine supplemented with dCF.3
DISCUSSION
The purine acquisition pathway of L. donovani is multifac-
eted and intertwined. Biochemical investigations (2–4, 35) and
bioinformatic analysis of genome sequences from several Leish-
mania species (36, 37) revealed that Leishmania parasites
accommodate four enzymes, HGPRT, XPRT, APRT, and aden-
FIGURE 9. PFGE of ⌬hgprt/⌬xprt[Ino/Hyp] chromosomes. Chromosomes
from wild type (first lane), ⌬aprt (second lane), ⌬hgprt/⌬xprt (third lane), and
two ⌬hgprt/⌬xprt[Ino/Hyp] suppressor clones (fourth and fifth lanes) were
separated by PFGE using a pulse time of 60 s and stained with ethidium
bromide (A). A Southern blot of the PFG depicted in A was probed with the
full-length L. donovani APRT gene (B). The arrows in A depict the extrachromo-
somal amplifications in the two ⌬hgprt/⌬xprt[Ino/Hyp] suppressors. The lad-
der consists of fractionated yeast markers obtained from Saccharomyces cer-
osine kinase, that are capable of assimilating host purines into
the parasite nucleotide pool. Functional studies demonstrated
that none of the four enzymes in this redundant pathway is, by
itself, absolutely critical for purine salvage by and growth of the
parasite (5–9). The subsequent isolation and characterization
evisiae strain YNN295.
of a conditionally lethal ⌬hgprt/⌬xprt double knock-out pro-
vided genetic proof that either HGPRT or XPRT (but not both)
APRT sequences were associated with the amplified ϳ200-
and ϳ275-kb elements (Fig. 9B). Detailed analysis of the
extrachromosomal elements and amplification events in these
two ⌬hgprt/⌬xprt[Ino/Hyp] suppressor mutants is currently
underway.
is crucial for purine salvage, whereas APRT and adenosine
kinase are functionally reiterative (10). Unlike wild type para-
sites, the ⌬hgprt/⌬xprt null mutant exhibits a highly restricted
growth phenotype under defined growth conditions in culture
and is virtually noninfectious in murine macrophages (10). In
this investigation, we extended our in vitro infectivity studies
with the ⌬hgprt/⌬xprt strain to mice, a conventional and well
tested rodent model for visceral leishmaniasis (13–16). The
results of these in vivo investigations revealed that the ability of
⌬hgprt/⌬xprt parasites to infect mice was dramatically dimin-
ished (Fig. 1). Although a considerable number of ⌬hgprt/⌬xprt
parasites persisted throughout the 4-week mouse infection, lib-
eration of persistent amastigotes from mice that were inocu-
lated with knock-out parasites revealed that the mechanism of
persistence did not involve reversion of the allelic replacements
that created the ⌬hgprt/⌬xprt strain. However, analysis of the
genetic events that contribute to the persistence phenotype was
APRT Overexpression Suppresses the Conditionally Lethal
Growth Phenotype of ⌬hgprt/⌬xprt parasites—To determine
whether APRT amplification and overexpression was the prin-
cipal mechanism that led to the suppressor phenotype in the
⌬hgprt/⌬xprt[Ino/Hyp] isolates (Figs. 2–4), APRT was intro-
duced into the ⌬hgprt/⌬xprt cells on an episomal plasmid by
transfection with [pXG-BSD-APRT] and selected for growth on
hypoxanthine in the absence of dCF and blasticidin. Several
clones that could utilize hypoxanthine in the absence of dCF
were isolated, and Western blot analysis of these cells revealed,
as expected, robust production of APRT protein in those
isolates compared with wild type and ⌬hgprt/⌬xprt parasites
(data not shown). Analysis of the growth phenotype of the
⌬hgprt/⌬xprt[pAPRT] transfectants in various purines clearly
indicated that overexpression of APRT from the amplified 3 J. M. Boitz and B. Ullman, personal observations.
18562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 24•JUNE 11, 2010

Suppression of a Lethal Phenotype in Leishmania
(Fig. 8). Nevertheless, this ϳ30-fold
amplification and overexpression of
APRT in the ⌬hgprt/⌬xprt[Ino/
Hyp] suppressor clones in all prob-
ability allows for sufficient salvage
of inosine and hypoxanthine to jus-
tify the relatively sluggish but con-
tinuous growth of the parasites
under these purine-defined condi-
tions (Fig. 2B) and for the ability to
efficiently infect macrophages (Fig.
3). Moreover, as proven by the
FIGURE 10. Comparison of growth phenotypes of wild type, ⌬hgprt/⌬xprt, and ⌬hgprt/⌬xprt[pXG-BSD-
growth phenotype of the ⌬hgprt/
⌬xprt[pAPRT] cell line, APRT over-
expression can also account for the
ability of the suppressor clones to
utilize adenine, adenosine, and
inosine, all of which are funneled to
APRT] parasites. The abilities of wild type, ⌬hgprt/⌬xprt, and ⌬hgprt/⌬xprt[pXG-BSD-APRT] promastigotes to
grow with various purine sources were evaluated after seeding the parasites at a density of 5 ϫ 104 para-
sites/ml in 24-well plates and allowing the parasites to proliferate until those growing under permissive con-
ditions reached late exponential phase. The parasites were enumerated by hemocytometer, and the data
presented are the averages and standard deviations of three separate determinations. Ade, adenine; Ado,
adenosine; Gua, guanine; Guo, guanosine; Hyp, hypoxanthine; Ino, inosine; Xan, xanthine; Xao, xanthosine.
impractical, because ⌬hgprt/⌬xprt parasites obtained from hypoxanthine through robust adenine aminohydrolase and
mice required expansion in permissive growth conditions that inosine hydrolytic activities (2, 7, 10, 43–45) (Fig. 10).
would presumably counterselect against genetic events that
The amplified copies of the APRT locus that occurred in
enabled growth under the nonpermissive conditions (absence two of the ⌬hgprt/⌬xprt[Ino/Hyp] suppressor clones are
of dCF) that occurred in vivo. localized to extrachromosomal elements of 200 and 275 kb,
To dissect the underlying mechanism for the prolonged respectively (Fig. 9). These amplicons appear to be linear in
survival of ⌬hgprt/⌬xprt parasites in vivo, we attempted to nature by their pulse time-dependent migration on contour-
isolate the progeny of axenically cultured ⌬hgprt/⌬xprt clamped homogeneous electric field gels (46). Leishmania spe-
parasites under controlled and well defined experimental cies exhibit considerable chromosomal plasticity in response to
conditions that enabled sustained growth under nonpermis- stress and are known to amplify both circular and linear chro-
sive conditions. Purine sources that did not permit ⌬hgprt/ mosomal elements in response to selective pressure (32, 33,
⌬xprt proliferation (10) were used to isolate clonal popula- 47–50). Although the L. donovani genome has not been
tions of parasites that had suppressed the conditional reported, the genome of L. infantum, a species phylogenetically
lethality of the ⌬hgprt/⌬xprt lesion. Several clones that grew akin to L. donovani, reveals that APRT is localized ϳ29 kb from
on hypoxanthine were obtained after two rounds of selection the end of chromosome 26 (36). Further genetic and biophysi-
in nonpermissive conditions. These ⌬hgprt/⌬xprt[Ino/Hyp] cal experiments on the amplicons in the ⌬hgprt/⌬xprt[Ino/
clones exhibited an expanded growth phenotype compared Hyp] suppressor clones are planned but are well beyond the
with that of the ⌬hgprt/⌬xprt knock-out and were capable of scope of this work.
proliferating continuously in either inosine, hypoxanthine, ade-
The mechanism of persistence in the few ⌬hgprt/⌬xprt
nine, or adenosine as the sole purine source and no longer parasites that survived the mouse infection is difficult to
dependent upon dCF in the culture medium (Fig. 2). Like the assess because the surviving parasites that emerged from the
⌬hgprt/⌬xprt null mutant, the ⌬hgprt/⌬xprt[Ino/Hyp] sup- harvested livers and spleens of the mice infected with the null
pressor lines could not utilize xanthine, guanine, or their cor- parasites were expanded in permissive medium, i.e. adenine
responding purine nucleosides as exogenous purine sources. plus dCF, that would counterselect against any suppressor
Further characterization of ⌬hgprt/⌬xprt[Ino/Hyp] clones by mechanism. Nevertheless, when a Western blot was performed
Southern and Western blotting revealed a ϳ30-fold amplifica- on persistent parasites from two separate populations isolated
tion and overexpression of the APRT locus in the suppressors from mice, a slight augmentation of APRT protein was
(Fig. 5). Previous kinetic analyses of both native and recombi- observed in ⌬hgprt/⌬xprt cells (Fig. 7). This elevated APRT
nant APRT from L. donovani indicated that the enzyme was level in the persistent parasite populations supports but does
specific for 6-aminopurines and did not recognize any of the not prove that APRT amplification event is a suppressor mech-
6-oxypurines, including hypoxanthine (23, 34). Examination of anism for ⌬hgprt/⌬xprt persistence in vivo.
the catalytic binding pockets of high resolution crystal struc-
The amplification of the APRT locus in the ⌬hgprt/⌬xprt[Ino/
tures of various APRT proteins also implied that 6-oxypurines Hyp] suppressors that were generated in vitro under controlled
would not be effective nucleobase substrates for these enzymes experimental circumstances and the discovery of persistent
(38–42). However, we have established in our studies using parasites from mice infected with ⌬hgprt/⌬xprt parasites
massive quantities of recombinant APRT that hypoxanthine strongly intimate that drug targeting of both HGPRT and
could serve as an inefficient substrate for the enzyme in vitro. XPRT is not a valid therapeutic paradigm, because resis-
Calculations of the catalytic efficiencies of adenine and hypox- tance, likely by an APRT amplification and/or perhaps some
anthine for the L. donovani APRT established that the 6-amino- other mechanism, will presumably arise. Because the virulence
purine was a more effective substrate by 6 orders of magnitude deficit of ⌬hgprt/⌬xprt parasites implies that hypoxanthine is
JUNE 11, 2010•VOLUME 285•NUMBER 24
JOURNAL OF BIOLOGICAL CHEMISTRY 18563

Suppression of a Lethal Phenotype in Leishmania
Myler, P. J., Stuart, K., and Ullman, B. (1999) J. Biol. Chem. 274,
34403–34410
the predominant purine source ultimately available for salvage
within L. donovani amastigotes, at least in mice, such an APRT
amplification and overexpression is unlikely to arise if a purine
interconversion enzyme downstream from HGPRT or XPRT is
targeted. Because the product of hypoxanthine phosphoribosy-
lation is IMP, the downstream enzymes that synthesize adeny-
late nucleotides from IMP, adenylosuccinate synthetase, and
adenylosuccinate lyase are presumably essential for the conver-
sion of HGPRT and XPRT products to adenylate nucleotides.
The functional role of adenylosuccinate synthetase and ade-
nylosuccinate lyase enzymes in L. donovani promastigotes and
amastigotes is tractable to genetic analysis, a project that has
now been initiated.
26. Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., pp. 9.31–9.57 and 18.60–18.66, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY
27. Zarella-Boitz, J. M., Rager, N., Jardim, A., and Ullman, B. (2004) Mol.
Biochem. Parasitol. 134, 43–51
28. Shih, S., Hwang, H. Y., Carter, D., Stenberg, P., and Ullman, B. (1998)
J. Biol. Chem. 273, 1534–1541
29. Galazka, J., Striepen, B., and Ullman, B. (2006) Mol. Biochem. Parasitol.
149, 223–230
30. Kidder, G. W., Dewey, V. C., and Nolan, L. L. (1977) Arch Biochem. Bio-
phys. 183, 7–12
31. Sillero, M. A., Socorro, S., Baptista, M. J., Del Valle, M., De Diego, A., and
Sillero, A. (2001) Eur. J. Biochem. 268, 3605–3611
32. Wilson, K., Beverley, S. M., and Ullman, B. (1992) Mol. Biochem. Parasitol.
55, 197–206
Acknowledgments—We thank Drs. Mark Slifka, Hans-Peter Raue´,
Ian J. Amanna, Jeff Gold, and Ann Kelly for generous assistance and
patient teaching, which made the macrophage and in vivo mouse
experiments possible. We thank Dr. Phillip Yates for help with the tail
vein injections.
33. Hanson, S., Beverley, S. M., Wagner, W., and Ullman, B. (1992) Mol. Cell.
Biol. 12, 5499–5507
34. Allen, T., Henschel, E. V., Coons, T., Cross, L., Conley, J., and Ullman, B.
(1989) Mol. Biochem. Parasitol. 33, 273–281
35. Krenitsky, T. A., Koszalka, G. W., Tuttle, J. V., Adamczyk, D. L., Elion,
G. B., and Marr, J. J. (1979) Adv. Exp. Med. Biol. 122B, 51–56
36. Berriman, M., Berriman, M., Ghedin, E., Hertz-Fowler, C., Blandin, G.,
Renauld, H., Bartholomeu, D. C., Lennard, N. J., Caler, E., Hamlin, N. E.,
Haas, B., Bo¨hme, U., Hannick, L., Aslett, M. A., Shallom, J., Marcello, L.,
Hou, L., Wickstead, B., Alsmark, U. C., Arrowsmith, C., Atkin, R. J., Bar-
ron, A. J., Bringaud, F., Brooks, K., Carrington, M., Cherevach, I., Chilling-
worth, T. J., Churcher, C., Clark, L. N., Corton, C. H., Cronin, A., Davies,
R. M., Doggett, J., Djikeng, A., Feldblyum, T., Field, M. C., Fraser, A.,
Goodhead, I., Hance, Z., Harper, D., Harris, B. R., Hauser, H., Hostetler, J.,
Ivens, A., Jagels, K., Johnson, D., Johnson, J., Jones, K., Kerhornou, A. X.,
Koo, H., Larke, N., Landfear, S., Larkin, C., Leech, V., Line, A., Lord, A.,
Macleod, A., Mooney, P. J., Moule, S., Martin, D. M., Morgan, G. W.,
Mungall, K., Norbertczak, H., Ormond, D., Pai, G., Peacock, C. S., Peter-
son, J., Quail, M. A., Rabbinowitsch, E., Rajandream, M. A., Reitter, C.,
Salzberg, S. L., Sanders, M., Schobel, S., Sharp, S., Simmonds, M., Simpson,
A. J., Tallon, L., Turner, C. M., Tait, A., Tivey, A. R., Van Aken, S., Walker,
D., Wanless, D., Wang, S., White, B., White, O., Whitehead, S., Wood-
ward, J., Wortman, J., Adams, M. D., Embley, T. M., Gull, K., Ullu, E.,
Barry, J. D., Fairlamb, A. H., Opperdoes, F., Barrell, B. G., Donelson, J. E.,
Hall, N., Fraser, C. M., Melville, S. E., and El-Sayed, N. M. (2005) Science
309, 416–422
REFERENCES
1. Berens, R. L., Krug, E. C., and Marr, J. J. (1995) in Biochemistry and Mo-
lecular Biology of Parasites (Marr, J. J., and Muller, M., eds) pp. 89–117,
Academic Press Ltd., San Diego, CA
2. Looker, D. L., Berens, R. L., and Marr, J. J. (1983) Mol. Biochem. Parasitol.
9, 15–28
3. Marr, J. J., Berens, R. L., and Nelson, D. J. (1978) Biochim. Biophys. Acta
544, 360–371
4. Tuttle, J. V., and Krenitsky, T. A. (1980) J. Biol. Chem. 255, 909–916
5. Hwang, H. Y., Gilberts, T., Jardim, A., Shih, S., and Ullman, B. (1996)
J. Biol. Chem. 271, 30840–30846
6. Hwang, H. Y., and Ullman, B. (1997) J. Biol. Chem. 272, 19488–19496
7. Boitz, J. M., and Ullman, B. (2006) Mol. Biochem. Parasitol. 148, 24–30
8. Iovannisci, D. M., Goebel, D., Allen, K., Kaur, K., and Ullman, B. (1984)
J. Biol. Chem. 259, 14617–14623
9. Iovannisci, D. M., and Ullman, B. (1984) Mol. Biochem. Parasitol. 12,
139–151
10. Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089
11. Robinson, N., Kaur, K., Emmett, K., Iovannisci, D. M., and Ullman, B.
(1984) J. Biol. Chem. 259, 7637–7643
37. Opperdoes, F. R., and Szikora, J. P. (2006) Mol. Biochem. Parasitol. 147,
193–206
12. Kidder, G. W., and Nolan, L. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76,
3670–3672
38. Phillíps, C. L., Ullman, B., and Brennan, R. G. (1996) Proteins 25, 510–513
39. Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J.
18, 3533–3545
13. Mullen, A. B., Baillie, A. J., and Carter, K. C. (1998) Antimicrob. Agents
Chemother. 42, 2722–2725
14. Wilson, M. E., Jeronimo, S. M., and Pearson, R. D. (2005) Microb. Pathog.
38, 147–160
40. Shi, W., Sarver, A. E., Wang, C. C., Tanaka, K. S., Almo, S. C., and
Schramm, V. L. (2002) J. Biol. Chem. 277, 39981–39988
41. Shi, W., Tanaka, K. S., Crother, T. R., Taylor, M. W., Almo, S. C., and
Schramm, V. L. (2001) Biochemistry 40, 10800–10809
15. Garg, R., and Dube, A. (2006) Indian J. Med. Res. 123, 439–454
16. Boitz, J. M., Yates, P. A., Kline, C., Gaur, U., Wilson, M. E., Ullman, B., and
Roberts, S. C. (2009) Infect. Immun. 77, 756–763
42. Silva, M., Silva, C. H., Iulek, J., Oliva, G., and Thiemann, O. H. (2004)
Biochim. Biophys. Acta 1696, 31–39
17. Goyard, S., Segawa, H., Gordon, J., Showalter, M., Duncan, R., Turco, S. J.,
and Beverley, S. M. (2003) Mol. Biochem. Parasitol. 130, 31–42
18. Dwyer, D. M. (1972) J. Parasitol. 58, 847–848
43. Koszalka, G. W., and Krenitsky, T. A. (1979) J. Biol. Chem. 254,
8185–8193
19. Holbrook, T. W., and Palczuk, N. C. (1975) Am. J. Trop. Med. Hyg. 24,
704–706
44. Shi, W., Schramm, V. L., and Almo, S. C. (1999) J. Biol. Chem. 274,
21114–21120
20. Debrabant, A., Joshi, M. B., Pimenta, P. F., and Dwyer, D. M. (2004) Int. J.
Parasitol. 34, 205–217
45. Cui, L., Rajasekariah, G. R., and Martin, S. K. (2001) Gene 280, 153–162
46. Sobral, B. W., and Atherly, A. G. (1989) Nucleic Acids Res. 17, 7359–7369
47. Ouellette, M., Fase-Fowler, F., and Borst, P. (1990) EMBO J. 9, 1027–1033
48. Wilson, K., Collart, F. R., Huberman, E., Stringer, J. R., and Ullman, B.
(1991) J. Biol. Chem. 266, 1665–1671
21. Iovannisci, D. M., and Ullman, B. (1983) J. Parasitol. 69, 633–636
22. Buffet, P. A., Sulahian, A., Garin, Y. J., Nassar, N., and Derouin, F. (1995)
Antimicrob. Agents Chemother. 39, 2167–2168
23. Allen, T., Hwang, H. Y., Wilson, K., Hanson, S., Jardim, A., and Ullman, B.
(1995) Mol. Biochem. Parasitol. 74, 99–103
49. Beverley, S. M., Coderre, J. A., Santi, D. V., and Schimke, R. T. (1984) Cell
38, 431–439
24. Allen, T. E., Hwang, H. Y., Jardim, A., Olafson, R., and Ullman, B. (1995)
Mol. Biochem. Parasitol. 73, 133–143
50. Ellenberger, T. E., and Beverley, S. M. (1989) J. Biol. Chem. 264,
15094–15103
25. Jardim, A., Bergeson, S. E., Shih, S., Carter, N., Lucas, R. W., Merlin, G.,
18564 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 24•JUNE 11, 2010

Metabolism:
Amplification of Adenine
Phosphoribosyltransferase Suppresses the
Conditionally Lethal Growth and Virulence
Phenotype of Leishmania donovani
Mutants Lacking Both
Hypoxanthine-guanine and Xanthine
Phosphoribosyltransferases
Jan M. Boitz and Buddy Ullman
J. Biol. Chem. 2010, 285:18555-18564.
doi: 10.1074/jbc.M110.125393 originally published online April 2, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M110.125393
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 48 references, 22 of which can be accessed free at
http://www.jbc.org/content/285/24/18555.full.html#ref-list-1